Investigation of structure and physical-chemical properties of proteins-enzymes. Mechanism of enzyme action, kinetic of enzymatic catalysis, role of cofactors and coenzyme vitamins in catalytic action of enzymes.

 

Characteristics of Enzymes

Enzymes are proteins that catalyze (i.e. accelerate) chemical reactions. In these reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. Almost all processes in a biological cell need enzymes in order to occur at significant rates. Since enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Like all catalysts, enzymes work by lowering the activation energy (ΔG‡) for a reaction, thus dramatically accelerating the rate of the reaction. Most enzyme reaction rates are millions of times faster than those of comparable uncatalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. Enzymes are known to catalyze about 4,000 biochemical reactions. Not all biochemical catalysts are proteins, since some RNA molecules called ribozymes also catalyze reactions.

http://www.youtube.com/watch?v=AFbPHlhI13g&feature=related

http://www.youtube.com/watch?v=AEsQxzeAry8&feature=related

http://www.youtube.com/watch?v=KED6BHVM97s&feature=related

 

Enzymes are protein molecules that are tailored to recognize and bind specific reactants and speed their conversion into products. These proteins are responsible for increasing the rates of all of the many thousand of reaction taking place inside cells.

All enzymatic proteins have several characteristics in common table 1.

Table 1: Characteristics of enzymes proteins

 

The rate of combination and release, known as the turnover number, lies near 1000 per second for most enzymes. Some enzymes have turnover numbers as small as 100 per second or as large as 10 million per second. As a result of enzyme turnover, a relatively small number of enzyme molecules can catalyze a large number of reactant molecules.

The part of an enzyme that combines with substrate molecule is the active site. In most enzymes the active site is located in a cavity or pocket on the enzyme surface, frequently within a cleft marking the boundary between two or more major domains. Within the cleft or pocket, amino acid side groups are situated to fit and bind parts of substrate molecules that are critical to the reaction catalyzed by the enzyme. The active site also separates substrate molecules from the surrounding solutions and place them in environments with unique characteristics, including partial or complete exclusion of water.

How Enzymes Lower the Energy of Activation

The mechanisms by which enzymes lower the energy of activation are still not totally understood. However, the mechanisms are believed to be directly or indirectly related to achievement of what is known as the transition state for a reaction. During any chemical interaction the reactants briefly enter a state in which old chemical bonds are incompletely broken and new ones are incompletely formed. In this transition state electron orbital assume intermediate positions between their locations in the reactants and their positions in the products. The transition state is highly unstable and can easily move in either direction with little change in energy – forward toward products ore back ward toward reactants. In effect, achievement of the transition state places a reacting system in a poised and precariously balanced position at the top of the activation energy barrier.

For example, in the transfer of a phosphate group from one molecule to another, a transition state is set up in which both molecules (shown as X and Y in Figure 2) link to the phosphate group a fraction of a second via transitory bonds (dotted lines). This unstable state can change readily in the direction of either products or unchanged reactants.

Enzymes as Biological Catalysts

In cells and organisms most reactions are catalyzed by enzymes, which are regenerated during the course of a reaction. These biological catalysts are physiologically important because they speed up the rates of reactions that would otherwise be too slow to support life. Enzymes increase reaction rates— sometimes by as much as one millionfold, but more typically by about one thousand fold. Catalysts speed up the forward and reverse reactions proportionately so that, although the magnitude of the rate constants of the forward and reverse reactions is are increased, the ratio of the rate constants remains the same in the presence or absence of enzyme. Since the equilibrium constant is equal to a ratio of rate constants, it is apparent that enzymes and other catalysts have no effect on the equilibrium constant of the reactions they catalyze.

Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, pH, and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins, making the meat easier to chew).

http://www.youtube.com/watch?v=cXLpxe6sPwI&feature=related

 

Most enzymes are much larger than the substrates they act on, and only a very small portion of the enzyme (around 3–4 amino acids) is directly involved in catalysis.

 

http://www.youtube.com/watch?v=0pJOFze055Y

 

The region that contains these catalytic residues, binds the substrate, and then carries out the reaction is known as the active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis. Some enzymes also have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction catalyzed.

Ribbon-diagram showing carbonic anhydrase II. The grey sphere is the zinc cofactor in the active site. Diagram drawn from PDB 1MOO.The activities of enzymes are determined by their three-dimensional structure. 

This binding can serve to increase or decrease the enzyme’s activity, providing a means for feedback regulation. Enzymes are catalysts. Most are proteins. (A few ribonucleoprotein enzymes have been discovered and, for some of these, the catalytic activity is in the RNA part rather than the protein part. Link to discussion of these ribozymes.)

Enzymes bind temporarily to one or more of the reactants of the reaction they catalyze. In doing so, they lower the amount of activation energy needed and thus speed up the reaction.

Examples:

·   Catalase. It catalyzes the decomposition of hydrogen peroxide into water and oxygen.

2H₂O₂ -> 2H₂O + O₂

One molecule of catalase can break 40 million molecules of hydrogen peroxide each second.

·   Carbonic anhydrase. It is found in red blood cells where it catalyzes the reaction

CO₂ + H₂O <-> H₂CO₃

It enables red blood cells to transport carbon dioxide from the tissues to the lungs. One molecule of carbonic anhydrase can process one million molecules of CO₂ each second.

Like all proteins, enzymes are made as long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a unique structure, which has unique properties. Individual protein chains may sometimes group together to form a protein complex. Most enzymes can be denatured—that is, unfolded and inactivated—by heating, which destroys the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible.

Specificity

Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity.

Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have “proof-reading” mechanisms. Here, an enzyme such as DNA polymerase catalyses a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases. Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes.

Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of different substrates. It has been suggested that this broad substrate specificity is important for the evolution of new biosynthetic pathways.

 “Lock and key” model

Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as “the lock and key” model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.

Induced fit model

 Diagrams to show the induced fit hypothesis of enzyme action.In 1958 Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site can be reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site, the amino acid side chains which make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site.

Providing an alternative pathway (e.g. temporarily reacting with the substrate to form an intermediate ES Complex which would be impossible in the absence of the enzyme).

Reducing the reaction entropy change by bringing substrates together in the correct orientation to react. Considering ΔH‡ alone overlooks this effect.

  Dynamics and function

Recent investigations have provided new insights into the connection between internal dynamics of enzymes and their mechanism of catalysis. An enzyme’s internal dynamics are described as the movement of internal parts (e.g. amino acids, a group of amino acids, a loop region, an alpha helix, neighboring beta-sheets or even entire domain) of these biomolecules, which can occur at various time-scales ranging from femtoseconds to seconds. Networks of protein residues throughout an enzyme’s structure can contribute to catalysis through dynamic motions. Protein motions are vital to many enzymes, but whether small and fast vibrations or larger and slower conformational movements are more important depends on the type of reaction involved. These new insights also have implications in understanding allosteric effects, producing designer enzymes and developing new drugs.

 

Cofactors and coenzymes

Role of Coenzymes

The functional role of coenzymes is to act as transporters of chemical groups from one reactant to another. The chemical groups carried can be as simple as the hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen carried by FAD; or they can be even more complex than the amine (-NH2) carried by pyridoxal phosphate.

Since coenzymes are chemically changed as a consequence of enzyme action, it is often useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different holoenzymes. In all cases, the coenzymes donate the carried chemical grouping to an acceptor molecule and are thus regenerated to their original form. This regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as a chemical catalyst, since (unlike the usual substrates, which are used up during the course of a reaction) coenzymes are generally regenerated.

 Enzyme Relative to Substrate Type

Enzymes also are generally specific for a particular steric configuration (optical isomer) of a substrate. Enzymes that attack D sugars will not attack the corresponding L isomer. Enzymes that act on L amino acids will not employ the corresponding D optical isomer as a substrate. The enzymes known as racemases provide a striking exception to these generalities; in fact, the role of racemases is to convert D isomers to L isomers and vice versa. Thus racemases attack both D and L forms of their substrate.

Enzyme cofactors

Many enzymes require the presence of an additional, nonprotein, cofactor.

·   Some of these are metal ions such as Zn²⁺ (the cofactor for carbonic anhydrase), Cu²⁺, Mn²⁺, K⁺, and Na⁺.

·   Some cofactors are small organic molecules called coenzymes. The B vitamins are precursors of coenzymes.

o                          thiamine (B1)

o                          riboflavin (B2) and

o                          nicotinamide

Coenzymes may be covalently bound to the protein part (called the apoenzyme) of enzymes as a prosthetic group. Others bind more loosely and, in fact, may bind only transiently to the enzyme as it performs its catalytic act.

Lysozyme: a model of enzyme action

A number of lysozymes are found iature; in human tears and egg white, for examples. The enzyme is antibacterial because it degrades the polysaccharide that is found in the cell walls of many bacteria. It does this by catalyzing the insertion of a water molecule at the position indicated by the red arrow. This hydrolysis breaks the chain at that point.

The bacterial polysaccharide consists of long chains of alternating amino sugars:

·   N-acetylglucosamine (NAG)

·   N-acetylmuramic acid (NAM)

These hexose units resemble glucose except for the presence of the side chains containing amino groups.
Lysozyme is a globular protein with a deep cleft across part of its surface. Six hexoses of the substrate fit into this cleft.

·   With so many oxygen atoms in sugars, as many as 14 hydrogen bonds form between the six amino sugars and certain amino acid R groups such as Arg-114, Asn-37, Asn-44, Trp-62, Trp-63, and Asp-101.

·   Some hydrogen bonds also form with the C=O groups of several peptide bonds.

·   In addition, hydrophobic interactions may help hold the substrate in position.

As for lysozyme itself, binding of the substrate induces a small (~0.75Å) movement of certain amino acid residues so the cleft closes slightly over its substrate. So the “lock” as well as the “key” changes shape as the two are brought together. (This is sometimes called “induced fit”.)

The amino acid residues in the vicinity of rings 4 and 5 provide a plausible mechanism for completing the catalytic act. Residue 35, glutamic acid (Glu-35), is about 3Å from the -O- bridge that is to be broken. The free carboxyl group of glutamic acid is a hydrogen ion donor and available to transfer H⁺ to the oxygen atom. This would break the already-strained bond between the oxygen atom and the carbon atom of ring 4.

Now having lost an electron, the carbon atom acquires a positive charge. Ionized carbon is normally very unstable, but the attraction of the negatively-charged carboxyl ion of Asp-52 could stabilize it long enough for an -OH ion (from a spontaneously dissociated water molecule) to unite with the carbon. Even at pH 7, water spontaneously dissociates to produce H⁺ and OH^– ions. The hydrogen ion (H⁺) left over can replace that lost by Glu-35.

In either case, the chain is broken, the two fragments separate from the enzyme, and the enzyme is free to attach to a new location on the bacterial cell wall and continue its work of digesting it.

Cofactors

Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules to be bound for activity. Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds, (e.g., flavin and heme). Organic cofactors (coenzymes) are usually prosthetic groups, which are tightly bound to the enzymes that they assist. These tightly-bound cofactors are distinguished from other coenzymes, such as NADH, since they are not released from the active site during the reaction.

 An example of an enzyme that contains a cofactor is carbonic anhydrase, and is shown in the ribbon diagram above with a zinc cofactor bound in its active site. These tightly-bound molecules are usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions.

 Enzymes that require a cofactor but do not have one bound are called apoenzymes. An apoenzyme together with its cofactor(s) is called a holoenzyme (i.e., the active form). Most cofactors are not covalently attached to an enzyme, but are very tightly bound. However, organic prosthetic groups can be covalently bound (e.g., thiamine pyrophosphate in the enzyme pyruvate dehydrogenase).

 Coenzymes

 Space-filling model of the coenzyme NADH Coenzymes are small molecules that transport chemical groups from one enzyme to another. Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins, this is when these compounds cannot be made in the body and must be acquired from the diet. The chemical groups carried include the hydride ion (H-) carried by NAD or NADP+, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine.

 Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 700 enzymes are known to use the coenzyme NADH.

 

Coenzymes are usually regenerated and their concentrations maintained at a steady level inside the cell: for example, NADPH is regenerated through the pentose phosphate pathway and S-adenosylmethionine by methionine adenosyltransferase.

Factors Affecting Enzyme Action

The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH (left graph) and temperature (right graph), its activity decreasing at values above and below that point. This is not surprising considering the importance of

·   tertiary structure (i.e. shape) in enzyme function and

·   noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.

Examples:

·   the protease pepsin works best as a pH of 1-2 (found in the stomach) while

·   the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents).

Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Without the unionized -COOH group of Glu-35 and the ionized -COO^– of Asp-52, the catalytic action of lysozyme would cease.

 

Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve (red arrow in right-hand graph above) reflects the general effect of increasing temperature on the rate of chemical reactions (graph at left). The descending portion of the curve above (blue arrow) reflects the loss of catalytic activity as the enzyme molecules become denatured at high temperatures.

Regulation of Enzyme Activity

Several mechanisms work to make enzyme activity within the cell efficient and well-coordinated.

 Anchoring enzymes in membranes

Many enzymes are inserted into cell membranes, for examples,

·   the plasma membrane

·   the membranes of mitochondria and chloroplasts

·   the endoplasmic reticulum

·   the nuclear envelope

These are locked into spatial relationships that enable them to interact efficiently.

Inactive precursors

Enzymes, such as proteases, that can attack the cell itself are inhibited while within the cell that synthesizes them. For example, pepsin is synthesized within the chief cells (in gastric glands) as an inactive precursor, pepsinogen. Only when exposed to the low pH outside the cell is the inhibiting portion of the molecule removed and active pepsin produced.

http://www.youtube.com/watch?v=duN73LFWNlo&feature=related

Feedback Inhibition

 If the product of a series of enzymatic reactions, e.g., an amino acid, begins to accumulate within the cell, it may specifically inhibit the action of the first enzyme involved in its synthesis (red bar). Thus further production of the enzyme is halted.

Precursor Activation

The accumulation of a substance within a cell may specifically activate (blue arrow) an enzyme that sets in motion a sequence of reactions for which that substance is the initial substrate. This reduces the concentration of the initial substrate.

In the case if feedback inhibition and precursor activation, the activity of the enzyme is being regulated by a molecule which is not its substrate. In these cases, the regulator molecule binds to the enzyme at a different site than the one to which the substrate binds. When the regulator binds to its site, it alters the shape of the enzyme so that its activity is changed. This is called an allosteric effect.

·   In feedback inhibition, the allosteric effect lowers the affinity of the enzyme for its substrate.

·   In precursor activation, the regulator molecule increases the affinity of the enzyme in the series for its substrate.If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase – by transcribing and translating the necessary gene(s) – and so can begin to metabolize the sugar.

Е. coli also has a mechanism which regulates enzyme synthesis by controlling translation of a needed messenger RNA..

Factors Affecting Enzymes

Thermodynamics

Main aticles: Activation energy, Thermodynamic equilibrium, and Chemical equilibrium.

Diagram of a catalytic reaction, showing the energy niveau at each stage of the reaction. The substrates usually need a large amount of energy to reach the transition state, which then decays into the end product. The enzyme stabilizes the transition state, reducing the energy needed to form this species and thus reducing the energy required to form products.As all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. Usually, in the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly. However, in the absence of the enzyme, other possible uncatalyzed, “spontaneous” reactions might lead to different products, because in those conditions this different product is formed faster.

Furthermore, enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to “drive” a thermodynamically unfavorable one. For example, the hydrolysis of ATP is often used to drive other chemical reactions.

Enzymes catalyze the forward and backward reactions equally. They do not alter the equilibrium itself, but only the speed at which it is reached. For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants.

 (in tissues; high CO2 concentration)

 (in lungs; low CO2 concentration)

Nevertheless, if the equilibrium is greatly displaced in one direction, that is, in a very exergonic reaction, the reaction is effectively irreversible. Under these conditions the enzyme will, in fact, only catalyze the reaction in the thermodynamically allowed direction.

Kinetics

 Mechanism for a single substrate enzyme catalyzed reaction. The enzyme (E) binds a substrate (S) and produces a product (P).Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are obtained from enzyme assays. In 1913 Leonor Michaelis and Maud Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis-Menten kinetics. Their work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today.

 Michaelis-Menton Kinetics

In typical enzyme-catalyzed reactions, reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration. Consequently, each enzyme molecule catalyzes the conversion to product of many reactant molecules. In biochemical reactions, reactants are commonly known as substrates. The catalytic event that converts substrate to product involves the formation of a transition state, and it occurs most easily at a specific binding site on the enzyme. This site, called the catalytic site of the enzyme, has been evolutionarily structured to provide specific, high-affinity binding of substrate(s) and to provide an environment that favors the catalytic events. The complex that forms, when substrate(s) and enzyme combine, is called the enzyme substrate (ES) complex. Reaction products arise when the ES complex breaks down releasing free enzyme.

 Between the binding of substrate to enzyme, and the reappearance of free enzyme and product, a series of complex events must take place. At a minimum an ES complex must be formed; this complex must pass to the transition state (ES*); and the transition state complex must advance to an enzyme product complex (EP). The latter is finally competent to dissociate to product and free enzyme. The series of events can be shown thus:

E + S <—> ES <—> ES* <—> EP <—> E + P

The kinetics of simple reactions like that above were first characterized by biochemists Michaelis and Menten. The concepts underlying their analysis of enzyme kinetics continue to provide the cornerstone for understanding metabolism today, and for the development and clinical use of drugs aimed at selectively altering rate constants and interfering with the progress of disease states. The Michaelis-Menten equation is a quantitative description of the relationship among the rate of an enzyme- catalyzed reaction [v1], the concentration of substrate [S] and two constants, Vmax and Km (which are set by the particular equation). The symbols used in the Michaelis-Menton equation refer to the reaction rate [v1], maximum reaction rate (Vmax), substrate concentration [S] and the Michaelis-Menton constant (Km).

  The Michaelis-Menten equation can be used to demonstrate that at the substrate concentration that produces exactly half of the maximum reaction rate, i.e.,1/2 Vmax, the substrate concentration is numerically equal to Km. This fact provides a simple yet powerful bioanalytical tool that has been used to characterize both normal and altered enzymes, such as those that produce the symptoms of genetic diseases. Rearranging the Michaelis-Menton equation leads to:

From this equation it should be apparent that when the substrate concentration is half that required to support the maximum rate of reaction, the observed rate, v1, will, be equal to Vmax divided by 2; in other words, v1 = [Vmax/2]. At this substrate concentration Vmax/v1 will be exactly equal to 2, with the result that:

[S](1) = Km

The latter is an algebraic statement of the fact that, for enzymes of the Michaelis-Menten type, when the observed reaction rate is half of the maximum possible reaction rate, the substrate concentration is numerically equal to the Michaelis-Menten constant. In this derivation, the units of Km are those used to specify the concentration of S, usually Molarity.

 The Michaelis-Menten equation has the same form as the equation for a rectangular hyperbola; graphical analysis of reaction rate (v) versus substrate concentration [S] produces a hyperbolic rate plot.

  Plot of substrate concentration versus reaction velocity

 

 The key features of the plot are marked by points A, B and C. At high substrate concentrations the rate represented by point C the rate of the reaction is almost equal to Vmax, and the difference in rate at nearby concentrations of substrate is almost negligible. If the Michaelis-Menten plot is extrapolated to infinitely high substrate concentrations, the extrapolated rate is equal to Vmax. When the reaction rate becomes independent of substrate concentration, or nearly so, the rate is said to be zero order. (Note that the reaction is zero order only with respect to this substrate. If the reaction has two substrates, it may or may not be zero order with respect to the second substrate). The very small differences in reaction velocity at substrate concentrations around point C (near Vmax) reflect the fact that at these concentrations almost all of the enzyme molecules are bound to substrate and the rate is virtually independent of substrate, hence zero order. At lower substrate concentrations, such as at points A and B, the lower reaction velocities indicate that at any moment only a portion of the enzyme molecules are bound to the substrate. In fact, at the substrate concentration denoted by point B, exactly half the enzyme molecules are in an ES complex at any instant and the rate is exactly one half of Vmax. At substrate concentrations near point A the rate appears to be directly proportional to substrate concentration, and the reaction rate is said to be first order.

 Inhibition of Enzyme Catalyzed Reactions

 To avoid dealing with curvilinear plots of enzyme catalyzed reactions, biochemists Lineweaver and Burk introduced an analysis of enzyme kinetics based on the following rearrangement of the Michaelis-Menten equation:

[1/v] = [Km (1)/ Vmax[S] + (1)/Vmax]

 Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax and an intercept on the ordinate at 1/Vmax.

 

A Lineweaver-Burk Plot

 An alternative linear transformation of the Michaelis-Menten equation is the Eadie-Hofstee transformation:

v/[S] = -v [1/Km] + [Vmax/Km]

 and when v/[S] is plotted on the y-axis versus v on the x-axis, the result is a linear plot with a slope of -1/Km and the value Vmax/Km as the intercept on the y-axis and Vmax as the intercept on the x-axis.

Both the Lineweaver-Burk and Eadie-Hofstee transformation of the Michaelis-Menton equation are useful in the analysis of enzyme inhibition. Since most clinical drug therapy is based on inhibiting the activity of enzymes, analysis of enzyme reactions using the tools described above has been fundamental to the modern design of pharmaceuticals. Well- known examples of such therapy include the use of methotrexate in cancer chemotherapy to semi-selectively inhibit DNA synthesis of malignant cells, the use of aspirin to inhibit the synthesis of prostaglandins which are at least partly responsible for the aches and pains of arthritis, and the use of sulfa drugs to inhibit the folic acid synthesis that is essential for the metabolism and growth of disease-causing bacteria. In addition, many poisons, such as cyanide, carbon monoxide and polychlorinated biphenols (PCBs). produce their life- threatening effects by means of enzyme inhibition.

 The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis-Menten complex in their honor. The enzyme then catalyzes the chemical step in the reaction and releases the product.

Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v).Enzymes can catalyze up to several million reactions per second. For example, the reaction catalyzed by orotidine 5′-phosphate decarboxylase will consume half of its substrate in 78 million years if no enzyme is present. However, when the decarboxylase is added, the same process takes just 25 milliseconds. Enzyme rates depend on solution conditions and substrate concentration. Conditions that denature the protein abolish enzyme activity, such as high temperatures, extremes of pH or high salt concentrations, while raising substrate concentration tends to increase activity. To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen. This is shown in the saturation curve, shown on the right. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the maximum velocity (Vmax) of the enzyme, all enzyme active sites are saturated with substrate, and the amount of ES complex is the same as the total amount of enzyme.

 

 

Inhibitor Type

Specifically at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate.

 Vmax is unchanged; Km, as defined by [S] required for 1/2 maximal activity, is increased.

http://www.youtube.com/watch?v=0pJOFze055Y

Noncompetitive Inhibitor

 Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate.

 Km appears unaltered; Vmax is decreased proportionately to inhibitor concentration.

 

 

Uncompetitive Inhibitor

 Binds only to ES complexes at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate.

 Apparent Vmax decreased; Km, as defined by [S] required for 1/2 maximal activity, is decreased.

 The hallmark of all the reversible inhibitors is that when the inhibitor concentration drops, enzyme activity is regenerated. Usually these inhibitors bind to enzymes by non-covalent forces and the inhibitor maintains a reversible equilibrium with the enzyme. The equilibrium constant for the dissociation of enzyme inhibitor complexes is known as KI:

KI = [E][I]/[E–I–complex]

The importance of KI is that in all enzyme reactions where substrate, inhibitor and enzyme interact, the normal Km and or Vmax for substrate enzyme interaction appear to be altered. These changes are a consequence of the influence of KI on the overall rate equation for the reaction. The effects of KI are best observed in Lineweaver-Burk plots.

Probably the best known reversible inhibitors are competitive inhibitors, which always bind at the catalytic or active site of the enzyme. Most drugs that alter enzyme activity are of this type. Competitive inhibitors are especially attractive as clinical modulators of enzyme activity because they offer two routes for the reversal of enzyme inhibition, while other reversible inhibitors offer only one. First, as with all kinds of reversible inhibitors, a decreasing concentration of the inhibitor reverses the equilibrium regenerating active free enzyme. Second, since substrate and competitive inhibitors both bind at the same site they compete with one another for binding .Raising the concentration of substrate (S), while holding the concentration of inhibitor constant, provides the second route for reversal of competitive inhibition. The greater the proportion of substrate, the greater the proportion of enzyme present in competent ES complexes.

As noted earlier, high concentrations of substrate can displace virtually all competitive inhibitor bound to active sites. Thus, it is apparent that Vmax should be unchanged by competitive inhibitors. This characteristic of competitive inhibitors is reflected in the identical vertical-axis intercepts of Lineweaver-Burk plots, with and without inhibitor.

Since attaining Vmax requires appreciably higher substrate concentrations in the presence of competitive inhibitor, Km (the substrate concentration at half maximal velocity) is also higher, as demonstrated by the differing negative intercepts on the horizontal axis in panel B.

 Analogously, panel C illustrates that noncompetitive inhibitors appear to have no effect on the intercept at the x-axis implying that noncompetitive inhibitors have no effect on the Km of the enzymes they inhibit. Since noncompetitive inhibitors do not interfere in the equilibration of enzyme, substrate and ES complexes, the Km’s of Michaelis-Menten type enzymes are not expected to be affected by noncompetitive inhibitors, as demonstrated by x-axis intercepts in panel C. However, because complexes that contain inhibitor (ESI) are incapable of progressing to reaction products, the effect of a noncompetitive inhibitor is to reduce the concentration of ES complexes that can advance to product. Since Vmax = k2[Etotal], and the concentration of competent Etotal is diminished by the amount of ESI formed, noncompetitive inhibitors are expected to decrease Vmax, as illustrated by the y-axis intercepts in panel C.

 A corresponding analysis of uncompetitive inhibition leads to the expectation that these inhibitors should change the apparent values of Km as well as Vmax. Changing both constants leads to double reciprocal plots, in which intercepts on the x and y axes are proportionately changed; this leads to the production of parallel lines in inhibited and uninhibited reactions.

 Inhibition

Competitive inhibitors bind reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor. Substrate and inhibitor compete for the enzyme.Main article: Enzyme inhibitor

Enzyme reaction rates can be decreased by various types of enzyme inhibitors.

Reversible inhibitors

In competitive inhibition the inhibitor binds to the substrate binding site (figure right, top, thus preventing substrate from binding (EI complex). Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of folic acid and this drug are shown in the figure to the right bottom.

Non-competitive inhibition

In order to do its work, an enzyme must unite – even if ever so briefly – with at least one of the reactants. In most cases, the forces that hold the enzyme and its substrate are noncovalent, an assortment of:

·   hydrogen bonds

·   ionic interactions and hydrophobic interactions

 

Most of these interactions are weak and especially so if the atoms involved are farther than about one angstrom from each other. So successful binding of enzyme and substrate requires that the two molecules be able to approach each other closely over a fairly broad surface. Thus the analogy that a substrate molecule binds its enzyme like a key in a lock.

 

 

This requirement for complementarity in the configuration of substrate and enzyme explains the remarkable specificity of most enzymes. Generally, a given enzyme is able to catalyze only a single chemical reaction or, at most, a few reactions involving substrates sharing the same general structure.

The necessity for a close, if brief, fit between enzyme and substrate explains the phenomenon of competitive inhibition.

It catalyzes the oxidation (by the removal of two hydrogen atoms) of succinic acid (a). If one adds malonic acid to cells, or to a test tube mixture of succinic acid and the enzyme, the action of the enzyme is strongly inhibited. This is because the structure of malonic acid allows it to bind to the same site on the enzyme (b). But there is no oxidation so no speedy release of products. The inhibition is called competitive because if you increase the ratio of succinic to malonic acid in the mixture, you will gradually restore the rate of catalysis. At a 50:1 ratio, the two molecules compete on roughly equal terms for the binding (=catalytic) site on the enzyme.

Non-competitive inhibitors can bind either to the active site, or to other parts of the enzyme far away from the substrate-binding site. Moreover, non-competitive inhibitors bind to the enzyme-substrate (ES) complex and to the free enzyme. Their binding to this site changes the shape of the enzyme and stops the active site binding substrate(s). Consequently, since there is no direct competition between the substrate and inhibitor for the enzyme, the extent of inhibition depends only on the inhibitor concentration and will not be affected by the substrate concentration.

Control activity

There are five main ways that enzyme activity is controlled in the cell.

Regulation of Enzyme Activity

While it is clear that enzymes are responsible for the catalysis of almost all biochemical reactions, it is important to also recognize that rarely, if ever, do enzymatic reactions proceed in isolation. The most common scenario is that enzymes catalyze individual steps of multi-step metabolic pathways, as is the case with glycolysis, gluconeogenesis or the synthesis of fatty acids. As a consequence of these lock- step sequences of reactions, any given enzyme is dependent on the activity of preceding reaction steps for its substrate.

In humans, substrate concentration is dependent on food supply and is not usually a physiologically important mechanism for the routine regulation of enzyme activity. Enzyme concentration, by contrast, is continually modulated in response to physiological needs. Three principal mechanisms are known to regulate the concentration of active enzyme in tissues:

 1. Regulation of gene expression controls the quantity and rate of enzyme synthesis.

  2. Proteolytic enzyme activity determines the rate of enzyme degradation.

3. Covalent modification of preexisting pools of inactive proenzymes produces active enzymes.

Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell in response to changes in the cell’s environment. This form of gene regulation is called enzyme induction and inhibition. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule. Another example are enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause drug interactions.

Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic reticulum and the Golgi apparatus and used by a different set of enzymes as a source of energy in the mitochondrion, through β-oxidation.

Enzymes can be regulated by inhibitors and activators. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps allocate materials and energy economically, and prevents the manufacture of excess end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms.

Enzymes can be regulated through post-translational modification. This can include phosphorylation, myristoylation and glycosylation. For example, in the response to insulin, the phosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or degradation of glycogen and allows the cell to respond to changes in blood sugar. Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. This type of inactive precursor to an enzyme is known as a zymogen.

Some enzymes may become activated when localized to a different environment (eg. from a reducing (cytoplasm) to an oxidising (periplasm) environment, high pH to low pH etc). For example, hemagglutinin of the influenza virus undergoes a conformational change once it encounters the acidic environment of the host cell vesicle causing its activation.

Allosteric modulation

Allosteric enzymes change their structure in response to binding of effectors. Modulation can be direct, where the effector binds directly to binding sites in the enzyme, or indirect, where the effector binds to other proteins or protein subunits that interact with the allosteric enzyme and thus influence catalytic activity.

In addition to simple enzymes that interact only with substrates and inhibitors, there is a class of enzymes that bind small, physiologically important molecules and modulate activity in ways other than those described above. These are known as allosteric enzymes; the small regulatory molecules to which they bind are known as effectors. Allosteric effectors bring about catalytic modification by binding to the enzyme at distinct allosteric sites, well removed from the catalytic site, and causing conformational changes that are transmitted through the bulk of the protein to the catalytically active site(s).

The hallmark of effectors is that when they bind to enzymes, they alter the catalytic properties of an enzyme’s active site. Those that increase catalytic activity are known as positive effectors. Effectors that reduce or inhibit catalytic activity are negative effectors.

Most allosteric enzymes are oligomeric (consisting of multiple subunits); generally they are located at or near branch points in metabolic pathways, where they are influential in directing substrates along one or another of the available metabolic paths. The effectors that modulate the activity of these allosteric enzymes are of two types. Those activating and inhibiting effectors that bind at allosteric sites are called heterotropic effectors. (Thus there exist both positive and negative heterotropic effectors.) These effectors can assume a vast diversity of chemical forms, ranging from simple inorganic molecules to complex nucleotides such as cyclic adenosine monophosphate (cAMP). Their single defining feature is that they are not identical to the substrate.

In many cases the substrate itself induces distant allosteric effects when it binds to the catalytic site. Substrates acting as effectors are said to be homotropic effectors. When the substrate is the effector, it can act as such, either by binding to the substrate-binding site, or to an allosteric effector site. When the substrate binds to the catalytic site it transmits an activity-modulating effect to other subunits of the molecule. Often used as the model of a homotropic effector is hemoglobin, although it is not a branch-point enzyme and thus does not fit the definition on all counts.

There are two ways that enzymatic activity can be altered by effectors: the Vmax can be increased or decreased, or the Km can be raised or lowered. Enzymes whose Km is altered by effectors are said to be K-type enzymes and the effector a K-type effector. If Vmax is altered, the enzyme and effector are said to be V-type. Many allosteric enzymes respond to multiple effectors with V-type and K-type behavior. Here again, hemoglobin is often used as a model to study allosteric interactions, although it is not strictly an enzyme.

In the preceding discussion we assumed that allosteric sites and catalytic sites were homogeneously present on every subunit of an allosteric enzyme. While this is often the case, there is another class of allosteric enzymes that are comprised of separate catalytic and regulatory subunits. The archetype of this class of enzymes is cAMP-dependent protein kinase (PKA), whose mechanism of activation is illustrated in the Figure below. The enzyme is tetrameric, containing two catalytic subunits and two regulatory subunits, and enzymatically inactive. When intracellular cAMP levels rise, one molecule of cAMP binds to each regulatory subunit, causing the tetramer to dissociate into one regulatory dimer and two catalytic monomers. In the dissociated form, the catalytic subunits are fully active; they catalyze the phosphorylation of a number of other enzymes, such as those involved in regulating glycogen metabolism. The regulatory subunits have no catalytic activity.

 

Representative pathway for the activation of cAMP-dependent protein kinase (PKA). In this example glucagon binds to its’ cell-surface receptor, thereby activating the receptor. Activation of the receptor is coupled to the activation of a receptor-coupled G-protein (GTP-binding and hydrolyzing protein) composed of 3 subunits. Upon activation the a-subunit dissociates and binds to and activates adenylate cyclase. Adenylate cylcase then converts ATP to cyclic-AMP (cAMP). The cAMP thus produced then binds to the regulatory subunits of PKA leading to dissociation of the associated catalytic subunits. The catalytic subunits are inactive until dissociated from the regulatory subunits. Once released the catalytic subunits of PKA phosphorylate numerous substrate using ATP as the phosphate donor.

Factors Affecting Enzyme Action

The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH (left graph) and temperature (right graph), its activity decreasing at values above and below that point. This is not surprising considering the importance of

·                     tertiary structure (i.e. shape) in enzyme function and noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.

Examples:

·                     the protease pepsin works best as a pH of 1-2 (found in the stomach) while

·                     the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents).

Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Without the unionized -COOH group of Glu-35 and the ionized -COO^– of Asp-52, the catalytic action of lysozyme would cease.

Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve (red arrow in right-hand graph above) reflects the general effect of increasing temperature on the rate of chemical reactions (graph at left). The descending portion of the curve above (blue arrow) reflects the loss of catalytic activity as the enzyme molecules become denatured at high temperatures.

Precursor Activation

The accumulation of a substance within a cell may specifically activate (blue arrow) an enzyme that sets in motion a sequence of reactions for which that substance is the initial substrate. This reduces the concentration of the initial substrate.

In the case if feedback inhibition and precursor activation, the activity of the enzyme is being regulated by a molecule which is not its substrate. In these cases, the regulator molecule binds to the enzyme at a different site than the one to which the substrate binds. When the regulator binds to its site, it alters the shape of the enzyme so that its activity is changed. This is called an allosteric effect.

·                     In feedback inhibition, the allosteric effect lowers the affinity of the enzyme for its substrate.

·                     In precursor activation, the regulator molecule increases the affinity of the enzyme in the series for its substrate.

Regulation of Enzyme Synthesis

The four mechanisms described above regulate the activity of enzymes already present within the cell.

What about enzymes that are not needed or are needed but not present?

Here, too, control mechanisms are at work that regulate the rate at which new enzymes are synthesized. Most of these controls work by turning on – or off – the transcription of genes.

If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase – by transcribing and translating the necessary gene(s) – and so can begin to metabolize the sugar.

E. coli also has a mechanism which regulates enzyme synthesis by controlling translation of a needed messenger RNA.

The Role of Ezymes in Biological Reactions

The laws of thermodymatics apply to chemical reactions anywhere in the universe. No reactions can violate the rules of thermodymatics. All reaction must proceed to a level of minimum energy and maximum entropy or have a favorable balance between the two. Enzyme simply increase the rate (rate is equal to the number of reactant molecules converted to product per unit of time) at which spontaneous reactions take place. Enzymes cannot make a reaction occur that would not proceed spontaneously without the enzyme. The same principle apply to reversible reaction. Enzymes do not alter the equilibrium point of reversible reaction.

Enzyme and Activation Energy

Activation energy is the energy barrier over which the molecules in a system must be raised for a reaction to take place (Figure 1).

This condition is analogous to a rock resting in a depression at the top of a hill. As long as the rock remains undisturbed, it will not spontaneously roll downhill unless activation energy is applied to the rock. Spontaneous movement over the barrier occurs because molecules, unlike the rock are in constant motion at temperatures above absolute zero. Although the average amount of movement, or kinetic energy, is below the amount required for activation, some molecular collision may raise a number of molecules to the energy level required for the reaction to proceed. The higher the activating barrier, the fewer the molecules that will proceed over the energy barrier per unit time.

Factors Affecting Enzyme Activity

A number of external factors affect the activity of enzymes in speeding conversion of reactants to products. These factors, including variations in the concentration for substrate molecules, temperature, and pH, speed or slow enzymatic activity in highly characteristic patterns.

Substrate Concentration

Enzymes react distinctively to alteration in the concentration of reacting molecules. At very low substrate concentration, collisions between enzyme and substrate molecules are infrequent and reaction proceeds slowly. As the substrate concentration increases, there reaction rate initially increases proportionately as collisions between enzyme molecules and reactants become more frequent Figure 3. When the enzymes begin to approach the maximum rate at which they can combine with reactants and release products, the effects of increasing substrate concentration diminish. At the point at which the enzymes are cycling as rapidly as possible, further increases in substrate concentration have no effect on there reaction rate. At this point the enzyme is saturated and the reaction remains at the saturation level.

If the reaction reaches a point at which further increases in reactants have no effect in increasing the rate of the reaction, then there is a good chance that the reaction is catalyzed by an enzyme. Uncatalyzed reactions, in contrast, increase the rate rate of the reaction almost indefinitely as the concentration of reactants increases.

The fact that enzymes combine briefly with their reactants makes them susceptible to inhibition by unreactive molecules that resemble the substrate. The inhibiting molecules can combine with the active site of the enzyme but tend to remain bound without change, blocking access by the normal substrate. As a result, the rate of there reaction slows. If the concentration of the inhibitor becomes high enough, the reaction may stop completely. Inhibition of this type is called competitive because the inhibitor competes with the normal substrate for binding to the active site.

Some inhibitors interfere with enzyme-catalyzed reactions by combining with enzymes at locations outside the active site. These inhibitors, rather than reducing accessibility of the active site to the substrate, cause changes in folding conformation that reduce the ability of the enzyme to lower the activation energy. Because such inhibitors do no directly compete for binding to the active site, their pattern of inhibition is called noncompetitive. Some poisons or toxins exert their damaging effects by acting as enzyme inhibitors. For example, the action of cyanide and carbon monoxide as poisons depends on their ability to inhibit enzyme important the utilization of oxygen in cellular respiration. Poisons and toxins typically act irreversibly by combining so strongly with enzymes, either covalently or nocovalently, that the inhibition is essentially permanent. Some irreversible poisons, rather than combining with the enzyme, destroy enzyme activity by chemically modifying critical amino acid side groups.

The cell has built in mechanisms to control directly both enzyme concentration and activity. First cells are able to regulate whether an enzyme is present at all. This type of control regulates protein synthesis and will be discussed in a later chapter. Cells also have ways to control the level of activity of enzymes that have already been synthesized and are present in the cell.

Ioncompetitive inhibition, a molecule binds to an enzyme but not at the active site. The other binding site is called the allosteric site (allo – other and steric -structure or space). The molecule that binds to the allosteric site is an inhibitor because it causes a change in the 3-dimensional structure of the enzyme that prevents the substrate from binding to the active site. In cells inhibition usually reversible; that is the inhibitor isn’t permanently bound to the enzyme. Irreversible inhibition of enzymes also occurs, due to the presence of a poison. For example, penicillin cause the death of bacteria due to irreversible inhibition of an enzyme needed to form the bacterial cell wall. In humans, hydrogen cyanide irreversibly bind to a very important enzyme (cytochrome oxidase) present in all cells, and this accounts for its lethal effect on the body.

The activity of almost every enzyme is a cell is regulated by feedback inhibition. Feedback inhibition is an example of common biological control mechanism called negative feedback. Just as high temperature will cause furnace to shut off, in a similar manner the product of an enzyme can inhibit a enzyme reaction. When the product is in abundance, it binds competitively with its enzyme’s active site; as the product is used up, inhibition is reduced and more product can be produced. In this way the concentration of the product is always controlled within a certain range.

Most enzymatic pathways are also regulated by feedback inhibition, but in these cases the end product of the pathway binds at an allosteric site on the first enzyme of the pathway. This binding shuts down the pathway, and not more product id produced. The reaction series converting theronine to isoleucine is a classic example of allosteric regulation. Five enzymes acting in sequence catalyze the pathway. The final product of the sequence, isoleucine, acts as an inhibitor of the first enzyme of the pathway, threonine deaminase. As the pathway produces isoleucine, any molecules made in excess of cell requirements combine reversibly with threonine deaminase at a location outside the active site. The combination converts threonine deaminase to the T state and inhibits its ability to combine with threonine. The pathway is then turned off. If the concentration of isoleucine later falls as a result of its use in cell synthesis, isoleucine releases from the threonine deaminase enzymes, converting them to the R state in which they have high affinity of the substrate, conversion of threonine to isoleucine takes place.

 Activation of an allosteric enzyme by an activator is another form of feedback inhibition. Combination of the activator and the allosteric site cause a conformational change in the active site permitting substrate binding and the reaction will be caltalyzed.

 Enzyme Regulation

REACTIONS AND ENZYMES

Energy releasing processes, ones that “generate” energy, are termed exergonic reactions. Reactions that require energy to initiate the reaction are known as endergonic reactions. All natural processes tend to proceed in such a direction that the disorder or randomness of the universe increases (the second law of thermodynamics).

Time-energy graphs of an exergonic reaction (top) and endergonic reaction (bottom). Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Oxidation/Reduction

Biochemical reactions in living organisms are essentially energy transfers. Often they occur together, “linked”, in what are referred to as oxidation/reduction reactions. Reduction is the gain of an electron. Sometimes we also have H ions along for the ride, so reduction also becomes the gain of H. Oxidation is the loss of an electron (or hydrogen). In oxidation/reduction reactions, one chemical is oxidized, and its electrons are passed (like a hot potato) to another (reduced, then) chemical. Such coupled reactions are referred to as redox reactions. The metabolic processes glycolysis, Kreb’s Cycle, and Electron Transport Phosphorylation involve the transfer of electrons (at varying energy states) by redox reactions.

Passage of electrons from compound A to compound B. When A loses its electrons it is oxidized; when B gains the electrons it is reduced.

Oxidation/reduction via an intermediary (energy carrier) compound, in this case NAD⁺. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Anabolism is the total series of chemical reactions involved in synthesis of organic compounds. Autotrophs must be able to manufacture (synthesize) all the organic compounds they need. Heterotrophs can obtain some of their compounds in their diet (along with their energy). For example humans can synthesize 12 of the 20 amino acids, we must obtain the other 8 in our diet. Catabolism is the series of chemical reactions that breakdown larger molecules. Energy is released this way, some of it can be utilized for anabolism. Products of catabolism can be reassembled by anabolic processes into new anabolic molecules.

Enzymes allow many chemical reactions to occur within the homeostasis constraints of a living system. Enzymes function as organic catalysts. A catalyst is a chemical involved in, but not changed by, a chemical reaction. Many enzymes function by lowering the activation energy of reactions. By bringing the reactants closer together, chemical bonds may be weakened and reactions will proceed faster than without the catalyst.

The use of enzymes can lower the activation energy of a reaction (E_a). Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Enzymes can act rapidly, as in the case of carbonic anhydrase (enzymes typically end in the -ase suffix), which causes the chemicals to react 107 times faster than without the enzyme present. Carbonic anhydrase speeds up the transfer of carbon dioxide from cells to the blood. There are over 2000 known enzymes, each of which is involved with one specific chemical reaction. Enzymes are substrate specific. The enzyme peptidase (which breaks peptide bonds in proteins) will not work on starch (which is broken down by human-produced amylase in the mouth).

Enzymes are proteins. The functioning of the enzyme is determined by the shape of the protein. The arrangement of molecules on the enzyme produces an area known as the active site within which the specific substrate(s) will “fit”. It recognizes, confines and orients the substrate in a particular direction.

Space filling model of an enzyme working on glucose. Note the shape change in the enzyme (indicated by the red arrows) after glucose has fit into the binding or active site.

The induced fit hypothesis suggests that the binding of the substrate to the enzyme alters the structure of the enzyme, placing some strain on the substrate and further facilitating the reaction. Cofactors are nonproteins essential for enzyme activity. Ions such as K⁺ and Ca⁺² are cofactors. Coenzymes are nonprotein organic molecules bound to enzymes near the active site. NAD (nicotinamide adenine dinucleotide).

A cartoonish view of the formation of an enzyme-substrate complex. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Enzymatic pathways form as a result of the common occurrence of a series of dependent chemical reactions. In one example, the end product depends on the successful completion of five reactions, each mediated by a specific enzyme. The enzymes in a series can be located adjacent to each other (in an organelle or in the membrane of an organelle), thus speeding the reaction process. Also, intermediate products tend not to accumulate, making the process more efficient. By removing intermediates (and by inference end products) from the reactive pathway, equilibrium (the tendency of reactions to reverse when concentrations of the products build up to a certain level) effects are minimized, since equilibrium is not attained, and so the reactions will proceed in the “preferred” direction.

Negative feedback and a metabolic pathway. The production of the end product (G) in sufficient quantity to fill the square feedback slot in the enzyme will turn off this pathway between step C and D. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Temperature: Increases in temperature will speed up the rate of nonenzyme mediated reactions, and so temperature increase speeds up enzyme mediated reactions, but only to a point. When heated too much, enzymes (since they are proteins dependent on their shape) become denatured. When the temperature drops, the enzyme regains its shape. Thermolabile enzymes, such as those responsible for the color distribution in Siamese cats and color camouflage of the Arctic fox, work better (or work at all) at lower temperatures.

Concentration of substrate and product also control the rate of reaction, providing a biofeedback mechanism.

Activation, as in the case of chymotrypsin, protects a cell from the hazards or damage the enzyme might cause.

Changes in pH will also denature the enzyme by changing the shape of the enzyme. Enzymes are also adapted to operate at a specific pH or pH range.

Plot of enzyme activity as a function of pH for several enzymes. Note that each enzyme has a range of pH at which it is active as well as an optimal pH at which it is most active. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Allosteric Interactions may allow an enzyme to be temporarily inactivated. Binding of an allosteric effector changes the shape of the enzyme, inactivating it while the effector is still bound. Such a mechanism is commonly employed in feedback inhibition. Often one of the products, either an end or near-end product act as an allosteric effector, blocking or shunting the pathway.

Action of an allosteric inhibitor as a negative control on the action of an enzyme.

Competitive Inhibition works by the competition of the regulatory compound and substrate for the binding site. If enough regulatory compound molecules bind to enough enzymes, the pathway is shut down or at least slowed down. PABA, a chemical essential to a bacteria that infects animals, resembles a drug, sulfanilamide, that competes with PABA, shutting down an essential bacterial (but not animal) pathway.

 

Top: general diagram showing competitor in the active site normally occupied by the natural substrate; Bottom: specific case of succinate dehydrogenase and its natural substrate (succinate) and competitors (oxalate et al.).

 Noncompetitive Inhibition occurs when the inhibitory chemical, which does not have to resemble the substrate, binds to the enzyme other than at the active site. Lead binds to SH groups in this fashion. Irreversible Inhibition occurs when the chemical either permanently binds to or massively denatures the enzyme so that the tertiary structure cannot be restored. Nerve gas permanently blocks pathways involved ierve message transmission, resulting in death. Penicillin, the first of the “wonder drug” antibiotics, permanently blocks the pathways certain bacteria use to assemble their cell wall components.

The four mechanisms described above regulate the activity of enzymes already present within the cell.

What about enzymes that are not needed or are needed but not present?

Here, too, control mechanisms are at work that regulate the rate at which new enzymes are synthesized. Most of these controls work by turning on — or off — the transcription of genes.

If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase — by transcribing and translating the necessary gene(s) — and so can begin to metabolize the sugar.

 

REGULATION OF METABOLITE FLOW CAN BE ACTIVE OR PASSIVE

Enzymes that operate at their maximal rate cannot respond to an increase in substrate concentration, and can respond only to a precipitous decrease in substrate concentration. For most enzymes, therefore, the average intracellular concentration of their substrate tends to be close to the Km value, so that changes in substrate concentration generate corresponding changes in metabolite flux. Responses to changes in substrate level represent an important but passive means for coordinating metabolite flow and maintaining homeostasis in quiescent cells. However, they offer limited scope for responding to changes in environmental variables. The mechanisms that regulate enzyme activity in an active manner in response to internal and external signals are discussed below.

 

Metabolite Flow Tends to Be Unidirectional

Despite the existence of short-term oscillations in metabolite concentrations and enzyme levels, living cells exist in a dynamic steady state in which the mean concentrations of metabolic intermediates remain relatively constant over time. While all chemical reactions are to some extent reversible, in living cells the reaction products serve as substrates for—and are removed by—other enzyme-catalyzed reactions.

 

Many nominally reversible reactions thus occur unidirectionally. This succession of coupled metabolic reactions is accompanied by an overall change in free energy that favors unidirectional metabolite flow. The unidirectional flow of metabolites through a pathway with a large overall negative change in free energy is analogous to the flow of water through a pipe in which one end is lower than the other. Bends or kinks in the pipe simulate individual enzyme-catalyzed steps with a small negative or positive change in free energy. Flow of water through the pipe nevertheless remains unidirectional due to the overall change in height, which corresponds to the overall change in free energy in a pathway.

 

 

COMPARTMENTATION ENSURES METABOLIC EFFICIENCY& SIMPLIFIES REGULATION

In eukaryotes, anabolic and catabolic pathways that interconvert common products may take place in specific subcellular compartments. For example, many of the enzymes that degrade proteins and polysaccharides reside inside organelles called lysosomes. Similarly, fatty acid biosynthesis occurs in the cytosol, whereas fatty acid oxidation takes place within mitochondria.

Segregation of certain metabolic pathways within specialized cell types can provide further physical compartmentation. Alternatively, possession of one or more unique intermediates can permit apparently opposing pathways to coexist even in the absence of physical barriers. For example, despite many shared intermediates and enzymes, both glycolysis and gluconeogenesis are favored energetically. This cannot be true if all the reactions were the same. If one pathway was favored energetically, the other would be accompanied by a change in free energy G equal in magnitude but opposite in sign. Simultaneous spontaneity of both pathways results from substitution of one or more reactions by different reactions favored thermodynamically in the opposite direction. The glycolytic enzyme phosphofructokinase is replaced by the gluconeogenic enzyme fructose-1,6-bisphosphatase. The ability of enzymes to discriminate between the structurally similar coenzymes NAD+ and NADP+ also results in a form of compartmentation, since it segregates the electrons of NADH that are destined for ATP generation from those of NADPH that participate in the reductive steps in many biosynthetic pathways.