Chapter 10

Enzymes: Their Kinetics, Specificity and Regulation

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Chapter Outline
v Enzymes Biological catalysts that function in dilute, aqueous solutions under mild conditions (e.g., pH and low temperatures) to increase reaction rate Catalytic power: Ratio of catalyzed rate to uncatalyzed rate Specificity Regulation of catalysis s Enzyme levels and types regulated genetically s Inhibitors and activators modify enzyme activity v Non-protein components Cofactors: Inorganic molecules Coenzymes: Organic molecules: Often vitamins Prosthetic group: Firmly bound coenzyme Holoenzyme: Apoenzyme plus prosthetic group v Chemical kinetics n Rate law: v = -dA/dt = k[A] s k = rate constant s n = order of reaction Molecularity: Number of molecules that must simultaneously react s Unimolecular: n = 1: k = first order rate constant s Bimolecular: k = second order rate constant n = 2, or v = k[A][B] v Reaction rates Limited by activation barrier: Free energy needed to reach transition state -G/RT Arrhenius equation k = Ae Influenced by s Temperature s Catalysts v Enzyme kinetics Saturation: Zero-order kinetics at high [S] Michaelis-Menten: v = (Vmax[S])/(Km+[S]) s s s
Km = (k-1+k2)/ k1 Km = [S] when v = Vmax/2 Conditions applicable [S]initial > [Enzyme] pH, temperature, ionic strength, [enzyme] constant Initial rate measured [S] essentially equal to [So]

Chapter 10 . Enzymes: Their Kinetics, Specificity and Regulation

[P] essentially zero v Enzyme parameters One international unit: Amount to catalyze formation of one micromole of product in one minute Turnover number = kcat = k2 = Vmax/[ET] k1 sets upper limit on catalytic efficiency: Reaction can go no faster than rate at which E and S form ES v Plots Direct plot: v versus [S]: v = (Vmax [S])/(Km+[S]) s Rectangular hyperbola s Asymptotically approaches Vmax when [S] high s
Km = [S] when v = Vmax/2

Lineweaver-Burk (double-reciprocal): 1/v versus 1/[S]: 1/v = (Km/Vmax)(1/[S])+1/Vm s Linear s Slope = Km/Vmax s
y-intercept =1/Vmax

s x-intercept = -1/Km v Temperature dependence of enzyme-catalyzed reactions Below 50C: Q10: Ratio of activities at two temperatures 10 apart: For typical enzyme Q10=2 Above 50C: Typically enzyme denatures v Inhibition Reversible inhibition: Noncovalent s Competitive inhibition: Inhibitor and substrate compete for same binding site Vmax unchanged Km = Km(1+[I]/KI) Noncompetitive inhibition Pure noncompetitive inhibition: S and I bind at different sites Mixed noncompetitive inhibition: I binding influences S binding Irreversible inhibition: Covalent s Suicide substrate or Trojan Horse substrate s Site-specific affinity label v Bisubstrate reactions: E + A + B C AEB C PEQ C E + P + Q Sequential or single displacement s Random: Either substrate binds to enzyme, either product is released s Ordered: Leading substrate binds first followed by second substrate Ping-Pong or double-displacement: Leading substrate binds: Enzyme modified: Product released: Second substrate binds: Enzyme unmodified: Second product released v Catalytic biomolecules Enzymes: Proteins Abzymes: Antibodies Ribozymes: RNA v Enzyme regulation Approach to equilibrium Km of enzymes in the range of in vivo substrate concentrations Genetic controls Covalent modification Allosteric regulation Others s Zymogens: Proenzymes or zymogens: Activated by proteolysis Proinsulin: Insulin Chymotrypsinogen: Chymotrypsin Blood clotting factors: Serine protease cascade leading to fibrinogen to fibrin s Isozymes: Lactate dehydrogenase: A4, A3B1, A2B2, A1B3, B4 s
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Chapter 10 . Enzymes: Their Kinetics, Specificity and Regulation

s Modular proteins: cAMP-dependent protein kinase: R2C2 v General properties of regulatory proteins Kinetic properties s Do not follow simple Michaelis-Menten kinetics s Activity sigmoidal: Higher order dependence on substrate concentration s Cooperativity Allosteric inhibition Often regulated by activation Oligomeric organization Effectors alter distribution of conformational isomers v Cooperativity models Monod, Wyman, Changeux (1965): Symmetry model s Two conformations: T (tense or taut) and R (relaxed) s R state high affinity, T state lower affinity s Positive homotropic effectors: Substrate binding shifts equilibrium to R s Heterotropic effectors Positive effector: Binds to R state and shifts equilibrium toward R Negative effector: Binds to T state and shifts equilibrium toward T Koshland, Nemethy, Filmer (1966) Sequential model: Induced fit: S-binding induces conformational change v Glycogen Phosphorylase Structure s Homodimer: 846-amino acids per monomer s Glycogen binding site s Phosphate binding site: Phosphate is positive homotropic effector s Serine 14: Site of covalent phosphorylation of enzyme: Phosphoprotein active and insensitive to allosteric regulation s ATP/AMP binding site: Heterotropic effectors: ATP inhibits: AMP activates s Glucose-6-phosphate binding site: Negative heterotropic regulator s Caffeine: Allosteric inhibitor v Enzyme cascade Hormone binds to cell surface receptor releasing GDP from heterotrimeric G-protein GTP binds to G subunit causing it to dissociate as G:GTP complex G:GTP complex stimulates adenylyl cyclase s
GTPase activity produces inactive G:GDP complex

s G:GDP complex must return to membrane for reactivation Adenylyl cyclase produces cAMP cAMP binds to regulatory subunits of R2C2 cAMP-dependent protein kinase s R subunist dissociate s C subunits become active Protein kinase phosphorylates glycogen phosphorylase kinase Glycogen phosphorylase kinase phosphorylates serine 14 of glycogen phosphorylase Protein phosphates removed by phosphoprotein phosphatase I

Chapter Objectives
Michaelis-Menten enzyme kinetics One of the keys to understanding Michaelis-Menten enzyme kinetics is to remember the model and its assumptions. The enzyme-substrate complex is in rapid equilibrium with free substrate. Product formation involves a fast catalytic step followed by a slow release of product. The velocity, , is the initial velocity of the reaction measured immediately upon addition of substrate when product concentration is very small. Typically the total enzyme concentration is small relative to substrate concentration and is kept constant. The Michaelis-Menten equation is not a complicated equation and is easy to understand.

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k 2 [E T ][S] Vmax [S] = K m + [S] k 1 + k 2 + [S] k1

It is easy to remember that Km has units of concentration because the denominator of the Michaelis-Menten equation is the term Km + [S]. Vmax is equal to the product of k2 and [ET]. Vmax is reached when substrate concentration is large. How large? Large relative to Km. When the substrate concentration is large relative to Km the enzyme is saturated and essentially all of it is tied up as ES complex. The concentration of ES complex is equal to the total enzyme concentration and the rate therefore is equal to k2[ET]. The equation is an example of a rectangular hyperbola. In a plot of vs. [S], Vmax is approached asymptotically as [S] increases and Km is the substrate concentration that supports = Vmax/2. Graphical representation The Lineweaver-Burk double-reciprocal plot uses 1/ and 1/[S] as variables because they are linearly related giving rise to straight lines whose slopes and intercepts are easily determined. A minor disadvantage to double-reciprocal plots is that they use inverse space: as [S] increases, 1/[S] decreases. With this in mind it is easy to remember that the 1/ intercept is equal to 1/Vmax, because the intercept occurs at the smallest real value of 1/. Smaller values of 1/ occur only to the left of the 1/-axis, where 1/[S] is negative. (Negative concentrations do not exist in the real world.) The 1/[S] intercept is equal to -1/Km.

Enzymes

Enzymes are proteins that act as biological catalysts but unlike inorganic catalysts, enzymes exhibit incredible specificity and can be regulated in numerous ways. Know the factors controlling enzymatic activity including: product build-up and approach to equilibrium, availability of substrates and cofactors, regulation of enzyme production and degradation, covalent modification, allosteric regulation, biosynthesis of enzymes as zymogens, isozymes, and regulation by modulator proteins. Keep in mind that enzymes as proteins may be capable of assuming different conformations and that the substrate-binding site may undergo local conformational change upon substrate binding. Allosteric Regulation In order to understand allosteric regulation, you must first understand standard MichaelisMenten kinetics. Plots of versus [S] are hyperbolic, approaching Vmax as [S] becomes large and having Km defined as the substrate concentration where = 0.5Vmax. Allosteric proteins exhibit sigmoidal plots of versus [S]. Inhibitors or activators that bind at sites distinct from the substrate-binding site yet still influence catalysis may influence the shape of the sigmoidal curve.

Allosteric Models Be familiar with the two models for allosteric behavior: the symmetry model of Monod, Wyman, and Changeux; and, the sequential model of Koshland, Nemethy, and Filmer. The symmetry model postulates the existence of two conformational states, R (relaxed) and T (taut), which differ in their affinity for substrate with R having a higher affinity than T. Cooperativity arises because substrate binding shifts the R/T equilibrium towards the R state. Allosteric activators and inhibitors bind at specific sites distinct from the substrate-binding site but influence substrate binding by influencing the R/T equilibrium. Inhibitor binding favors the T state whereas activator binding favors the R state. In the sequential model, ligand-binding sites are in communication with each other such that the state of occupancy of a ligand-binding site influences the state of occupancy of other sites. Inhibitors Competitive inhibition occurs whenever an inhibitor competes with a substrate for the substrate binding site in a reversible manner. How will the presence of a competitive inhibitor affect enzyme kinetics? Clearly, at high [S], S will out-compete the inhibitor. Thus, Vmax is unaffected. The apparent Km is increased to Km(1 + [I]/KI). (The term [I]/KI is the inhibitor concentration normalized to the inhibitor dissociation constant, KI.)

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Chapter 10 . Enzymes: Their Kinetics, Specificity and Regulation

In noncompetitive inhibition, the inhibitor binds reversibly to a site different than the substrate binding site but inhibitor-bound enzyme is inactive. The easiest case to remember is pure noncompetitive inhibition. Here, I binds to its enzyme binding site without regard to the state of occupancy of the substrate binding site. In other words, E and ES bind I with equal affinity. The consequence is a decrease in the apparent Vmax to Vmax/(1 + [I]/KI); however, Km is unaffected. Mixed noncompetitive inhibition occurs when I binding to E and ES differ. Both Km and Vmax are affected. Bisubstrate reactions Single-displacement or sequential reactions occur as follows: A + B + E \ AEB \ PEQ \ E + P + Q The two substrates may bind in a specific order or in random order. Ping-Pong or double-displacement reactions proceed as follows: A + E \ EA \E'P \E' + P; E' + B \ E'B \EQ \E + Q

Problems and Solutions

1. According to the Michaelis-Menten equation, what is the /Vmax ratio when [S] = 4Km? Answer:

Vmax [S] K m + [S] V max 4K m 4 Vmax = , or K m + 4K m 5 = 0.8

When [S] = 4K m = Vmax

2. If Vmax = 100 mol/mLsec and Km = 2 mM, what is the velocity of the reaction when [S] = 20 mM?

Answer: For Vmax = 100 mol/sec and Km = 2 mM, [S] = 20 mM V [S] = max K m + [S]
mol 20 mM mL sec = 2 mM + 20 mM mol 100 20 mM mL sec = 22 mM mol = 91 mL sec

100

3. For a Michaelis-Menten reaction, k1 = 7 x 10 / Msec, k-1 = 1 x 10 /sec and k2 = 2 x 4 10 /sec. What are the values of Km?

Answer: When k1 = 7 x 107/Msec, k-1 = 1 x 103/sec, k2 = 2 x 104/sec

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Chapter 10 . Enzymes: Their Kinetics, Specificity and Regulation

k 1 + k 2 k1

K m = Michaelis - Menten constant = 1 10 3 = + 2 104

sec sec 7 10 7

M sec K m = 3.0 104 M

4. The following kinetic data were obtained for an enzyme in the absence of inhibitor (1), and in the presence of two different inhibitors (2) and (3) at 5 mM concentration. Assume [ET] is the same in each experiment. [S] (1) (2) (3) (mM) (mol/mLsec) (mol/mLsec) (mol/mLsec) 1 12 4.3 5.5 2 20 8 9 4 29 14 13 5 35 21 16 12 40 26 18 a. Determine Vmax and Km for the enzyme. b. Determine the type of inhibition and the KI for each inhibitor.

Answer: The data may be analyzed using reciprocal variables. For each [S] and corresponding , we will calculate 1/[S] and 1/. [S] (mM) 1 2 4 5 12 1/[S] v(1) 1/v(1) M-1 umol/mL sec mL sec/mol 1000 12 8.33E+04 500 20 5.00E+04 250 29 3.45E+04 200 35 2.86E+04 83 40 2.50E+04 v(2) umol/mL sec 4.3 8 14 21 26 1/v(2) v(3) 1/v(3) mL sec/mol umol/mL sec mL sec/mol 2.33E+05 5.5 1.82E+05 1.25E+05 9 1.11E+05 7.14E+04 13 7.69E+04 4.76E+04 16 6.25E+04 3.85E+04 18 5.56E+04

2.50E+05

2.00E+05 1.50E+05
1/v

1.00E+05 5.00E+04

0.00E+00 -400 -200 0 200 400 1/[S] 600 800 1000 1200

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Chapter 10 . Enzymes: Their Kinetics, Specificity and Regulation

Plots of 1/ vs. 1/[S] indicate straight lines given by (1) 1/ = 63.1(1/[S] ) + 1.96 x 104 (2) 1/ = 212.3(1/[S]) + 1.99 x 104 (3) 1/ = 137.4(1/[S)) + 4.38 x 104 In general, 1 Km 1 1 = + V max [S] Vmax Thus, the y-intercept is equal to 1/Vmax and the x-intercept is equal to -1/Km. (a) Condition No inhibitor 5 mM inhibitor (2) 5 mM inhibitor (3) Vmax (mol/mLsec) 51 50.3 22.8 Km (mM) 3.2 10.7 3.1 This is

(b) Inhibitor (2) increases the apparent Km of the enzyme without affecting Vmax. characteristic of a competitive inhibitor. In this case KI is calculated as follows [ I] K m,app = K m (1 + ) or KI 10.7 mM = 3.2 mM(1 + Solving for K I we find 5 mM KI )

5 mM = 2.35 mM 10 mM 1) ( 3.2 mM Inhibitor (3) decreases Vmax but leaves Km unchanged, an example of noncompetitive inhibition. In this case KI is calculated as follows 1 1 [ I] = (1 + ) or V max,app V max KI KI =
1 [I ] 1 = (1+ ) KI 22.8 51 Solving for K I we find KI = ( 5 mM 51 1) 22.8 = 4.04 mM

5. The following graphical patterns obtained from kinetic experiments have several possible interpretations depending on the nature of the experiment and the variables being plotted. Give at least two possibilities for each.

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Chapter 10 . Enzymes: Their Kinetics, Specificity and Regulation

1 _

1 _

1 _ [S]

1 _ [S]

1 _

1 _ [S]
Answer: In the top left graph, the results may be due to a competitive inhibitor. The line with the steeper slope is from enzyme kinetic measurements in the presence of an inhibitor whereas the other line is enzyme kinetic data without inhibitor. In competitive inhibition, Vmax, the reciprocal of the 1/ intercept, is independent of the presence of inhibitor. However, Km,app increases, as reflected in the decrease (in absolute magnitude) of the 1/[S] intercept which equals 1/Km,app. In competitive inhibition, I and S compete for the same enzyme-binding site. The apparent Km increases because, in the presence of inhibitor, higher concentrations of substrate are required to half-saturate the enzyme. The lines are described by the following equation: [ I] K m (1 + ) KI 1 1 1 = + [ S] V max Vmax An alternative interpretation of the data is that the inhibitor binds to and forms a complex with the substrate. In this case the equation 1 Km 1 1 = + V max [S] Vmax must be modified to take this inhibitor-substrate interaction into account. In this equation, [S] is the initial concentration of substrate available to the enzyme. The concentration of substrate used to construct the plots is the total substrate concentration, [ST]. Under normal conditions, [ST] = [S], but in this case [ST] = [S] + [SI] because some of the substrate is complexed to I. We can derive an expression for [S] as follows: From [S T ] = [S] + [SI], we have [S] = [S T ] [SI], and using KI = [SI] = [I][S] , we see that [SI] [I][S] KI

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Chapter 10 . Enzymes: Their Kinetics, Specificity and Regulation

Substituting [SI] above and solving for [S] we find, [S T ] [S] = [I] 1+ KI By substituting into 1 the equation, it becomes :

[I] K m (1 + ) KI 1 1 1 = + Vmax [S T ] Vmax This equation is identical to the case of competitive inhibition. The top right hand graph may be a case of pure, noncompetitive inhibition, which is described by the following equation [ I] [ I] K m (1 + (1 + ) ) KI KI 1 1 = + [S] Vmax V max In this case the inhibitor binds to the enzyme and to the enzyme-substrate complex with equal affinity. The binding of I to E has no effect on the binding of S to E. Thus, Km is unaffected because the inhibitor-free enzyme is capable of normal catalysis. Vmax is decreased to Vmax/(1 + [I]/KI). In effect, the inhibitor lowers the active enzyme concentration. The data can also be explained as the result of experiments conducted at two different enzyme concentrations. (In this case no inhibition whatsoever is involved.) Another possibility, involving an inhibitor, is that the inhibitor binds irreversibly to E and inactivates it. This is the same as lowering the total enzyme concentration. Finally, we may be looking at a case of random, single-displacement bisubstrate reaction. Binding of substrate A is not affected by binding of B and vice versa. The bottom, center graph may be an example of mixed noncompetitive inhibition in which the inhibitor I binds to both E and ES but unequally. In this case, I binds to ES with higher affinity (i.e., lower K'I) than to S. Vmax is decreased and Km,app is increased. Alternatively, this may be an example of a single-displacement bisubstrate mechanism characterized by the following equation

1 K AK B 1 1 KB 1 = ( KA + S m ) + (1 + m ) m V max [ A ] Vmax [ B] [ B]
6. Liver alcohol dehydrogenase (ADH) is relatively nonspecific and will oxidize ethanol or other alcohols, including methanol. Methanol oxidation yields formaldehyde, which is quite toxic, causing, among other things, blindness. Mistaking it for the cheap wine he usually prefers, my dog Clancy ingested about 50 mL of windshield washer fluid (a solution 50% in methanol). Knowing that methanol would be excreted eventually by Clancys kidneys if its oxidation could be blocked, and realizing that, in terms of methanol oxidation by ADH, ethanol would act as a competitive inhibitor, I decided to offer Clancy some wine. How much of Clancys favorite vintage (12% ethanol) must he consume in order to lower the activity of his ADH on methanol to 5% of its normal value if the Km values of canine ADH for ethanol and methanol are 1 millimolar and 10 millimolar, respectively? (The KI for ethanol in its role as competitive inhibitor of methanol oxidation by ADH is the same as its Km). Both the methanol and ethanol will quickly distribute throughout Clancys body fluids, which amount to about 15 L. Assume the densities of 50% methanol and the wine are both 0.9.

Answer: The Km values of alcohol dehydrogenase for ethanol and methanol are l mM and 10 mM. How many moles of methanol are in 50 mL of a 50% solution (v/v)? The solution was made by adding 25 mL methanol and adjusting the volume to 50 mL with water. This amount of methanol (i.e., 25 mL) weighs 25 mL x 0.9 g/mL = 22.5 g. The molecular composition of methanol is CH4O with a weight of 32 g/mol. Thus, Clancy consumed 22.5 g 32 g/mol) = 0.7 moles of methanol. Fortunately for him, he diluted it into 15 L of body fluid giving a final concentration of 0.7 moles

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15 L = 46.9 mM. This concentration is well above the Km of alcohol dehydrogenase for methanol at 10 mM. We expect the enzyme to function at V max 46.9 mM = 10 mM + 46.9 mM = 0.82V max Now, how much ethanol must Clancy consume to lower to 5% of 0.82 Vmax or to about .041 Vmax? V max [S ] = , or [ I] K m (1 + ) + [S ] KI V max 46.9 mM 0.041V max = [ Ethanol] 10 mM(1 + ) + 46.9 mM 1.0 mM Solving for [Ethanol], we find [Ethanol] = 109 mM
To raise his alcohol concentration to 109 mM, he must drink: 109 mM 15 L = 1.63 moles of ethanol. If Clancy gets his alcohol (ethanol) from wine, how much wine must he drink? The Mr of ethanol (C2H6O) is 46, so 1.63 moles represents: g = 75g ethanol. 1.63 mol 46 mol At a density of 0.9 g/mL, this represents 75 g pure ethanol = 12% X grams of wine 75g Grams of wine = = 625 g .12 625g = 694 mL of wine g 0.9 mL Solving for VT, VT= 694 mL of 12% ethanol. Clancy needs about one 750-mL bottle of wine.
7. List six general ways in which enzyme activity is controlled.

Answer: Enzyme activity may be controlled by: 1. Accumulation of product as the reaction approaches equilibrium. As substrate is converted to product, [P] increases. 2. Availability of substrates and cofactors. 3. Regulation of the amounts of enzyme synthesized or degraded by cells. 4. Covalent modifications catalyzed by modifying enzymes or converter enzymes may activate or inhibit an enzyme or alter its kinetic properties. 5. Allosteric regulation leading to either inhibition or activation. 6. Regulation by interaction with modulator proteins. 7. Activation or inactivation by proteolysis.
8. Why do you suppose proteolytic enzymes are often synthesized as inactive zymogens?

Answer: Proteolytic enzymes hydrolyze peptide bonds of proteins, the most diverse and abundant class of biopolymers. Clearly cells must take care in the biosynthesis of proteases to guard against inadvertent digestion of cellular proteins. Thus, proteolytic enzymes are typically produced as inactive zymogens that are activated only at the site at which their action is required.
9. Draw a Lineweaver-Burk plots for the following: A Monod-Wyman-Changeux allosteric K enzyme system, showing separate curves for the kinetic response in (1) the absence of any effectors; (2) the presence of allosteric activator A; and (3) the presence of allosteric

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inhibitor I. Also draw a similar set of curves for a Monod-Wyman-Changeux allosteric V enzyme system.

Answer: The Monod-Wyman-Changeux model considers a multimeric enzyme with n substrate binding sites. The enzyme can exist in two states, R for relaxed and T for taut or tense, that differ in their affinity for substrate. The R state has high affinity whereas, in the extreme case, the T state has no affinity for substrate. Under given conditions (such as temperature, pH, ionic strength, etc.) R and T are in equilibrium and the ratio of their concentrations is governed by an equilibrium constant. Cooperativity occurs because substrate preferentially binds to R thus shifting the R to T equilibrium towards R. In the absence of cooperativity a multisubunit enzyme would obey simple Michaelis-Menten kinetics with each substrate binding site acting independently. In the cooperative case, substrate binding leads to an increase in the population of high affinity sites, which manifests itself as a higher order dependence of velocity on substrate concentration. In a purely cooperative system, the dependence order will be equal to the number of substrate binding sites in the multimeric protein. Michaelis-Menten kinetics is governed by Vmax[S] v= Km + [S] Cooperativity is described by
Kmn + [S]n The cooperativity equation may be further generalized to take into account heterotropic interactions. Heterotropic interactions refer to binding of molecules at sites other than substratebinding sites that influence the R to T equilibrium. In an allosteric K enzyme system, heterotropic interactions will influence Km, the substrate concentration at which the system is functioning at half Vmax. A heterotropic inhibitor shifts the equilibrium towards the T state and thus increases the apparent Km whereas a heterotropic activator shifts the equilibrium towards the R state and thus decreases the apparent Km. In order to include heterotropic interactions we replace Km with the following: (1 + ) Km, app = Km (1 + ) v= Vmax[S]n

Where = [I]/KI and = [A]/KA, and [I] and [A] refer to inhibitor and activator concentrations, and KI and KA are inhibitor and activator dissociation binding constants. The expression for v becomes:
v= Vmax[S] n (1 + ) n (Km ) + [S] n (1 + )

In the Lineweaver-Burk plot shown below the solid line represents cooperative kinetics in the absence of both inhibitor and activator for n = 2.0. The dashed line (below) is cooperative kinetics in the presence of activator. The dotted line (above) is cooperative kinetics in the absence of inhibitor. The concentration at which the system reaches half-maximum velocity (i.e., 1/v = 2) is lower in the presence of activator and higher in the presence of inhibitor.

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9 8 7 6

1/v

5 4 3 2 1 0 0 1 2 3 4 5 6

1/[S]
In an allosteric V enzyme system, heterotropic interactions will influence Vmax; Km apparent does not change. The Michaelis-Menten equation may be used with Vmax being modified by the term (1 + )/1 + ) or (1 + ) Vmax [S] (1 + ) v= Km + [S]

14 12 10 1/v 8 6 4 2 0
-3 -1 1 3 5

1/[S]

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Chapter 10 . Enzymes: Their Kinetics, Specificity and Regulation

In the plot shown above the solid line is in the absence of inhibitor and activator, the dashed line (below) is in the presence of activator, and the dotted line (above) is in the presence of inhibitor.
10. In the Monod-Wyman-Changeux model for allosteric regulation, what values of L and relative affinities of R and T for A will lead activator A to exhibit positive homotropic effects? (That is, under what conditions will the binding of A enhance further A-binding, in the same manner that S-binding shows positive cooperativity?) Similarly, what values of L and relative affinities of R and T for I will lead inhibitor I to exhibit positive homotropic effects? (That is, under what conditions will the binding of I promote further I-binding?)

Answer: The term L is the equilibrium constant for R to T transition: [T ] R o , To , and L = o [R o ] Large values of L indicate that the T state is favored over the R state, thus [To]>[Ro]. In this situation, A will show positive homotropic binding. Because A binds preferentially to the R state, A-binding will favor a substantial increase in the concentration of the R state. For small values of L, the R state is favored over the T state, thus [To]<[Ro]. Inhibitor binding to the T state will show positive homotropic binding in this situation because I binds preferentially to the T state.
11. The cAMP formed by adenylyl cyclase (Figure 10.42) does not persist because 5phosphodiesterase activity prevalent in cells hydrolyzes cAMP to give 5'-AMP. Caffeine inhibits 5-phosphodiesterase activity. Describe the effects on glycogen phosphorylase activity that arise as a consequence of drinking lots of caffeinated coffee.

Answer: By inhibiting phosphodiesterase activity cAMP levels will slowly rise or will remain elevated for longer periods of time. This will prolong stimulation of cAMP-dependent protein kinase leading to persistent phosphorylation of glycogen phosphorylase kinase and in turn glycogen phosphorylase. Caffeine is an allosteric inhibitor of glycogen phosphorylase b, the unphosphorylated form of the enzyme that is relatively inactive and sensitive to allosteric regulation. (Caffeine can influence three physiological conditions: Functioning of adenosine receptors, intracellular calcium levels, and cyclic nucleotide levels. Caffeine is a competitive inhibitor of adenosine receptors and is effective at low levels. In contrast, intracellular calcium levels and cyclic nucleotide levels are increased by caffeine but only at high levels of caffeine.)
12. Enzymes have evolved such that their Km values (or K0.5 values) for substrate(s) are roughly equal to the in vivo concentration(s) of the substrate(s). Assume that glycogen phosphorylase is assayed at [Pi] K0.5 in the absence and presence of AMP or ATP. Estimate from Figure 10.38 the relative glycogen phosphorylase activity when (a) neither AMP or ATP is present, (b) AMP is present, and (c) ATP is present.

Answer: K0.5 is defined as the concentration of substrate at which the enzyme functions at 0.5Vmax. From Figure 10.38b we can estimate the location of K0.5 on the x-axis by measuring the distance to Vmax along the -axis, drawing a line parallel to the x-axis at a value of equal to 0.5Vmax, and, where this line intersects the -ATP curve (upper curve), drawing a perpendicular. The point of intersection of the perpendicular on the x-axis is equal to K0.5. The intersection of this perpendicular on the +ATP curve, is for = K0.5 in the presence of ATP. a. = 0.5Vmax in the absence of ATP and AMP (by definition). b. 0.85Vmax in the presence of AMP. c. 0.12Vmax in the presence of ATP.

Questions for Self Study

1. Enzymes have three distinctive features that distinguish them from chemical catalysts. What are they?

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2. Match the terms in the first column a. k in = k[A] b. d[P]/dt or -d[S]/dt c. d[P]/dt = -d[S]/dt d. k in = k[A][B] e. k-1/k1 f. (k-1 + k2)/k1 g. at [S] = h. [S] when v = Vmax/2 i. kcat j. 1/ when 1/[S] = 0

with the descriptions in the second column. 1. Km 2. Vmax 3. First-order rate constant 4. Michaelis constant 5. Vmax/ET 6. The velocity or rate of a reaction. 7. Enzyme:substrate dissociation constant 8. 1/Vmax 9. Equilibrium or steady state 10. Second-order rate constant

3. Fill in the blanks. The energy barrier preventing thermodynamically favorable reactions from occurring is known as the _____. In general, catalysts _____ this energy barrier by providing an alternate reaction pathway leading from substrate to product. The intermediate along this reaction pathway that has the highest free energy is known as the _____. 4. Answer True or False a. For competitive inhibition the apparent Km is larger than Km by the factor (1 + [I]/KI). _____ b. For noncompetitive inhibition the apparent Vmax is larger than Vmax by the factor (1 + [I]/KI). _____ c. A plot of Vmax versus temperature is linear for a typical enzyme. _____ d. Ordered, single-displacement reactions refer to bisubstrate reactions in which two substrates bind to the enzyme before catalysis occurs. _____ e. Enzyme modification occurs during double-displacement or Ping-Pong reaction. _____ 5. What is a ribozyme? What is an abzyme?

6. What is the essential difference between the "lock and key" hypothesis of enzyme substrate interaction and the "induced fit" hypothesis? 7. How are the proteins, trypsinogen, chymotrypsinogen, pepsinogen, procarboxypeptidase, proelastase, related to active enzymes? 8. Lactate dehydrogenase is a tetramer that catalyzes the interconversion of lactate and pyruvate. There are two different subunits, A and B that can interact to form either homotetramers or heterotetramers with different kinetic properties. Write the five possible tetrameric combinations. What kind of enzyme regulation does lactate dehydrogenase typify? 9. The enzyme aspartate transcarbamoylase catalyzes the condensation of carbamoyl phosphate and aspartic acid to form N-carbamoylasparate. A plot of initial velocity versus aspartate concentration is not a rectangular hyperbola as found for enzymes that obey Michaelis-Menten kinetics. Rather, the plot is sigmoidal. Further, CTP shifts the curve to the right along the substrate axis and makes the sigmoidal shape more pronounced. ATP shifts the curve to the left and makes the sigmoidal shape less pronounced. What kind of enzyme is aspartate transcarbamoylase and what roles do CTP and ATP play in enzyme activity? 10. Match the items in the left hand column with items in the right hand column. a. Symmetry Model 1. Second noncatalytic substrate binding site. b. Monod, Wyman, and Changeux 2. Substrate binding concentration dependence. c. K system 3. Negative effector. d. V system 4. Conformation change due to substrate binding. e. Koshland, Nemethy, and Filmer 5. K0.5 changes in response to effectors. 6. Vmax regulated. f. Induced fit g. Hill coefficient 7. Sequential model. 8. RoC To h. Increases [R conformation] i. Decreases [R conformation] 9. Two conformational states.

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j. Homotropic effector 10. Positive effector.

Answers
1. Enormous catalytic power, specificity, and the ability to be regulated. 2. a. 3; b. 6; c. 9; d. 10; e. 7; f. 1 or 4; g. 2; h. 1 or 4; i. 5; j. 8. 3. Energy of activation; lower; transition state. 4. a. T; b. F; c. F; d. T; e. T 5. A ribozyme is a catalytic RNA. An abzyme is a catalytic antibody.

6. The "lock and key" hypothesis depicts the enzyme as a rigid template into which the substrate fits. In the "induced fit" hypothesis enzymes are highly flexible, dynamic molecules and substrate binding modifies the shape of the enzymes active site. 7. These proteins are precursors of the enzymes, trypsin, chymotrypsin, pepsin, carboxypeptidase, and elastase. They are converted to active enzymes by hydrolysis of peptide bonds. 8. A4, A3B, A2B2, AB3, B4; isozymes. 9. Aspartate transcarbamoylase is a regulatory enzyme. CTP is a negative heterotropic effector and ATP is a positive heterotropic effector. 10. a. 8 or 9; b. 8 or 9; c. 5; d. 6; e. 7; f. 4; g. 2; h. 10; i. 3; j. 1.

Additional Problems
1. A researcher was studying the kinetic properties of -galactosidase using an assay in which onitrophenol--galactoside (ONPG), a colorless substrate, is converted to galactose and onitrophenolate, a brightly-colored, yellow compound. Upon addition of substrate to 0.25 mM substrate to a fixed amount of enzyme, o-nitrophenolate (ONP) production was monitored as a function of time by spectrophotometry at = 410 nm. The following data were collected: Time A410 nm (sec) 0 0.000 15 0.158 30 0.273 45 0.360 60 0.429 75 0.484 90 0.529 150 0.652 210 0.724 270 0.771 330 0.805 390 0.830 450 0.849 510 0.864 a. Convert A410 nm to concentration of o-nitrophenolate, [ONP], using = 3.76 mM-1cm-1 as the extinction coefficient. b. Plot [ONP] versus time. c. Determine the initial velocity of the reaction. d. Explain why the curve is nearly linear initially and later approaches a plateau.

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e. Describe how Km and Vmax for -galactosidase can be determined with additional experimentation. 2. Under what conditions can an enzyme assay be used to determine the relative amounts of an enzyme present? 3. For many reactions, it is often difficult to measure product formation, so coupled assays are often used. In a coupled enzyme assay, the activity of an enzyme is determined by measuring the activity of a second enzyme that uses as substrate the product of a reaction catalyzed by the first enzyme. The utility of coupled assays is that the product of the second enzyme is easy or convenient to measure. You are asked to design a coupled assay to measure the relative amounts of a particular enzyme in several samples. In qualitative terms with respect to Km and Vmax for both enzymes, explain how an appropriate coupled assay is set up. 4. In the case of a competitive inhibitor, explain how the inhibitor dissociation constant, KI, can be determined from enzyme kinetic data. 5. Is Km a good measure of the dissociation constant for substrate binding to enzyme? Explain. 6. Derive an expression for as a function of [S] for mixed, noncompetitive inhibition. 7. For certain inhibitor/enzyme combinations the presence of inhibitor decreases Vmax; however, it also decreases Km,app. This seems to suggest that in the presence of an inhibitor the remaining active enzyme's performance is improved since it saturates at a lower substrate concentration. Can you explain this paradox? 8. Enzyme activity may be controlled in several ways (see answer to question 1 above). It is clear that some of these regulatory mechanisms are effective nearly instantly whereas others are slowly activated. With respect to duration, certain controls are short-lived whereas others are long-lasting. Compare and contrast enzyme control mechanisms with respect to these parameters.

Abbreviated Answers
1a. The concentration of ONP, [ONP], is calculated using Beer's Law, A = Cl, where is the extinction coefficient, C is the concentration, and l is the path length (1 cm in this case). [ONP] = A410 nm/(l cm) Time (sec) 0 15 30 45 60 75 90 150 210 270 330 390 450 510 A410nm 0 0.158 0.273 0.36 0.429 0.484 0.529 0.652 0.724 0.771 0.805 0.83 0.849 0.864 [ONP] (mM) 0 0.042 0.073 0.096 0.114 0.129 0.141 0.173 0.193 0.205 0.214 0.221 0.226 0.23

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b. The plot of [ONP] versus time is shown below 0.250 0.200 [ONP] (mM) 0.150 0.100 0.050 0.000 0 100 200 300 Time (sec) c. The initial velocity is equal to the slope of a line drawn through the initial points as shown above and has a value of 0.00188 mM/sec. d. The enzyme is converting substrate to product and the curve is the time course of this process. The initial points are linear because the enzyme is obeying Michaelis-Menten kinetics. At later times, two things happen to cause the rate of product formation, given by the slope of a tangent drawn along the graph, to decrease: [S] decreases; and, [P] increases. The curve is approaching 2.5 mM, the initial value of substrate, indicating that nearly all of the initial substrate has been converted to product. e. By repeating the experiment at different initial substrate concentrations, the initial velocities at varying substrate concentrations can be measured. These data are used to determine Km and Vmax. 2. At substrate concentrations high relative to Km, an enzyme functions at or near Vmax. Vmax, given by Vmax = k2ET, is proportional to the total enzyme concentration. 3. The following reaction sequence represents a coupled enzyme assay. S P1 P2 We are interested in determining the relative amount of E1 by measuring the rate of production of P2. The concentration of the substrate for E1, namely S, must be large relative to Km1 in order for E1 to function at Vmax. Also, the rate at which E2 produces P2 must be constant. This requires that [P1] be at a steady-state concentration. The rate of change of [P1] is given by Vmax,E2 [P1] d[P1] = Vmax,E1 dt K m2 + [P1] For [P1] to be at steady state, [P1]steady state = d[P1] = 0, and solving for [P1] we find : dt which is positive only when
E1 E2

400

500

600

K m2 Vmax,E1 Vmax,E 2 Vmax,E1

Vmax,E2 > Vmax,E1 Thus, [S] must be high enough to saturate E1 and k2[E2,T] must be greater than Vmax,E1. 4. For competitive inhibition, Km,app = Km(1 + [I]/KI). By measuring Km,app at several different inhibitor concentrations and plotting Km,app against [I], the slope of the resulting straight line divided by the y-intercept (equal to Km) is 1/KI.

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5. Km and Kd are defined as follows: k + k2 k K m = 1 , and K D = 1 k1 k1

When k 1 is large relative to k 2 , K m K D 6. The following reactions occur for mixed, noncompetitive inhibition: E + S C ES C P; E + I C EI; ES + I C IES Binding of I to E and to ES are governed by: [E][I] [ES][I] ; K 'I = KI = [EI] [ IES] These equations can be rearranged to give: [E][I ] [ES][I] [EI] = ; [IES] = KI K' I The rate of change of [ES] is given by: d[ES] = k 2 [ES] k 1[ES] + k1[E][S] dt Using this equation and making the steady-state assumption we find that k [E][S] [ E][S] [ES] = 1 = k 2 + k 1 Km The total concentration of enzyme, ET, is given by: [E T ] = [E] + [ES] + [EI] + [ IES] Substituting the expressions for [ES], [EI], and [IES] above we find that: [E][S] [ E][ I] [ E][S ][I] [ET ] = [E] + + + , and solving for [E] we find : Km KI K m K 'I

[ ET ] K m (1 + [E] = (1 + [I] K 'I [I] )

) KI Km + [S ] [I] ) (1 + K 'I The rate of product formation is given by: [ E][S ] Km Finally, substituting the expression for [E] above we find: k 2 [E T ] [ S] [ I] (1 + ) Vmax,app[S] K 'I = = [ I] K m,app + [S] ) (1 + KI + [S] Km [ I] ) (1 + K 'I = k 2[ES ] = k 2 Clearly, Vmax,app is always less than Vmax. Km,app may be less than, equal to, or greater than Km depending on the magnitude of KI relative to K'I. 7. I binds to ES with greater affinity (lower dissociation constant) than to E. Therefore, the addition of I is expected to affect the E + S , ES equilibrium by shifting it to the right allowing more ES to form at lower [S].

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8. Variations in [product], [S], and [cofactors] have immediate consequences for enzyme activity. The same is true for allosteric regulation. Further, these controls last as long as the signals are present. Genetic controls, such as induction and repression leading to an increase or decrease in enzyme synthesis, are fast, although not immediate, and can be long lasting. Clearly, degradation is long lasting. However, a delicate balance between synthesis and degradation can lead to a very responsive system of control. Covalent modification may lead to rapid and long lasting changes in enzyme activity. Balancing the activity of modifying enzymes against enzymes that can reverse these modifications creates a sensitive system of enzyme control. Finally, zymogens and isozymes are examples of enzyme control mechanisms that can be extremely long lasting.

Summary
Enzymes are the catalysts of metabolism, allowing living systems to achieve kinetic control over the thermodynamic potential within organic reactions. Enzymes have three distinctive attributes that are essential to their biological purpose: enormous catalytic power, great selectivity in catalyzing only very specific reactions, and the ability to be regulated so that their activity is compatible with the momentary needs of the cell. Formally, enzymes are classified by a system of nomenclature based on the particular reaction they catalyze, although certain trivial names for enzymes often enjoy common usage. Enzymes may be simple proteins or they may be proteins complexed with nonprotein components called cofactors. Cofactors include metal ions and organic molecules known as coenzymes. An enzyme accelerates the rate of a process by lowering the thermodynamic barrier to reaction, known as the free energy of activation. The reactant in an enzyme-catalyzed reaction is called the substrate. The fact that graphs of enzyme activity as a function of substrate concentration show a limiting plateau or saturation level was the important clue that an enzyme (E) actually binds its substrate (S) to form an enzyme:substrate (ES) complex that can react to yield product (P). Enzyme kinetic analysis is based on the notion put forth by Michaelis and Menten that E and S reversibly interact, and ES can react to form P. Briggs and Haldane extended this model by assuming that the system E + S ,ES C P quickly reaches a steady state condition where d[ES]/dt = 0. The standard Michaelis-Menten equation for an enzymatic reaction is then derived: V [S] = max K m + [S] Vmax is the maximal velocity attained when [S] is so great as to not be rate-limiting; Km is a constant reflecting the relative affinity and reactivity of the enzyme-substrate interaction. Because graphs of versus [S] are not linear, the Michaelis-Menten expression is often rearranged to give equations that yield linear relationships between rate and substrate concentration. Because their activity is highly dependent on maintenance of their native protein structure, enzymes are very sensitive to pH and temperature. Enzyme inhibition has been an important means for investigating enzymes and their mode of action. Such studies have also contributed to human health through the science of pharmacology, since many drugs exert their action as inhibitors of enzymes. Some inhibitors act by competing with the substrate for binding to the active site of the enzyme, altering the apparent Km. Other inhibitors uniquely affect the apparent Vmax while still others affect both Km,app and Vmax,app. Inhibitors, which react covalently with the enzyme, can lead to irreversible inhibition of its activity. Most enzymatic reactions involve more than one substrate. These multi-substrate reactions can be analyzed kinetically to determine whether two (or more) substrates are bound before reaction occurs (so-called sequential or single-displacement reactions), or whether one substrate binds and reacts with the enzyme before the other substrate is bound (Ping-Pong or doubledisplacement reactions). Recent discoveries have broadened our view of biocatalysis. Certain RNA molecules have the essential features of enzymes: rate enhancement, catalytic turnover and specificity of reaction. This finding has important implications to our view of molecular or pre-biotic evolution. Further, biocatalysts in the form of antibodies have been tailor-made by using transition state analogs of a reaction as antigens. The antibodies elicited in response to these analogs act as biocatalysts by facilitating entry of the reactants into a reactive transition-state intermediate. The creation of designer enzymes has become a realistic possibility.

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Enzymes display an extraordinary specificity with regard to the substrates they act upon and the reaction they catalyze. Molecular recognition through structural complementarity is the basis of this specificity. The active sites of enzymes are pockets or clefts in the protein structure whose shape and charge distribution are complementary to the substrates own properties. In turn, substrate binding induces changes in protein conformation so that the substrate is embraced by the enzyme and catalytic groups are brought to bear. In effect, the enzyme:substrate complex mimics the transition state intermediate of the reaction. Regulation of enzyme activity can be achieved at several levels: enzyme synthesis or degradation, reversible covalent modifications catalyzed by converter enzymes, noncovalent interactions with cellular metabolites (allosteric regulation), zymogen activation, isozymes or modular proteins. Allosteric regulation occurs when regulatory metabolites modulate the activity of allosteric enzymes by binding at sites distinct from the active site. Such effectors may activate (positively regulate) or inhibit (negatively regulate) enzyme activity. Often, an allosteric enzyme catalyzes the first step in an essential metabolic pathway. The end product of the pathway may be a feedback inhibitor (negative regulator) of this allosteric enzyme. Allosteric enzymes are characterized by sigmoid versus [S] kinetic patterns. The sigmoid shape of such kinetic curves suggests that the enzymatic rate is proportional to [S]n, where n > 1. Allosteric enzymes are oligomeric and possess multiple ligand-binding sites. The Monod, Wyman and Changeux model for the behavior of allosteric proteins postulates that allosteric proteins are oligomers composed of identical subunits. The protein can exist in either of two conformational states, the T or taut state, and the R or relaxed state. The distribution of the protein between the two conformational states is characterized by the equilibrium constant, L: L = To/Ro. The different states have different affinities for the various ligands. Usually, it is assumed that the substrate S binds only to the R state; the positive allosteric effector A binds only to the R state; and the negative allosteric effector I binds only to the T state. Given these parameters, the sigmoid rate response of allosteric enzymes to substrate concentration and the effects exerted by allosteric effectors are understood. The system described above is called a K system, because the substrate concentration giving halfmaximal velocity, defined as K0.5 , varies, while V remains constant. In the V system of allosteric control, R and T states have the same affinity for S, but the R state is catalytically active, while the T state is not. A and I still differ in their relative affinities for R and T. Both the K and the V systems assume that the oligomeric allosteric protein undergoes a concerted conformational transition so that all subunits have identical conformations at the same time. The Koshland, Nemethy and Filmer model rests on the induced fit hypothesis. That is, via tightly linked subunit interactions, the binding of ligand to one subunit can influence the ligandbinding affinity of neighboring subunits. Therefore, an oligomeric protein could pass through a sequential series of conformations with differing ligand affinities, and different subunits may not necessarily have the same conformation at the same time. Glycogen phosphorylase is a homodimeric protein that catalyzes the catabolism of glycogen into glucose-1-phosphate. The enzyme is inhibited by two allosteric inhibitors, ATP and glucose6-phosphate, which both function as negative heterotropic effectors by decreasing the affinity of Pi. AMP acts as a positive heterotropic effector by binding to the enzyme at the ATP-binding site. AMP binding not only blocks ATP binding but it increases the enzyme's affinity for Pi. These allosteric regulators all function on phosphorylase b. Phosphorylase a, a more active form of the enzyme, is relatively insensitive to allosteric regulation. The physical basis for the difference in these two forms is the presence of a phosphate group on serine 14. The phosphate group may be removed by phosphoprotein phosphatase I and reattached by glycogen phosphorylase kinase, an enzyme that is itself activated by phosphorylation. These covalent modifications are the result of an enzymatic cascade that is initiated with hormone binding to cell surface receptors, transduced into release of a GTP-binding protein that stimulates adenylyl cyclase, which in turn produces cAMP that stimulates cAMP-dependent protein kinase leading to phosphorylation of glycogen phosphorylase kinase.

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