NEW METHOD OF DETERMINING pK VALUES OF CATALYTIC GROUPS 85 2 3 4 5 6 7 −1.0 −0.5 0.0 0.5 1.0 pK es1 pK es2 pH ∆log V max /∆pH 2 3 4 5 6 7 −1.0 −0.5 0.0 0.5 1.0 pK e1 pK e2 pH ∆log (V * max /K s * )/ ∆pH 2 3 4 5 6 7 −0.50 −0.25 0.00 0.25 0.50 pK es1 pK es2 pK e1 pK e2 pH ∆(−log K s * )/∆pH ( a ) ( b ) ( c ) Figure 6.4. Variation in the slope of the (a)logV max ,(b)logV max /K s and (c) −log K s versus pH plots as a function of pH. 86 pH DEPENDENCE OF ENZYME-CATALYZED REACTIONS Consider the expression for the hydrogen ion dependence of the K s of an enzyme-catalyzed reaction: K ∗ s = K s 1 +[H + ]/K e1 + K e2 /[H + ] 1 +[H + ]/K es1 + K es2 /[H + ] = K s K es1 K e1 [H + ] 2 + K e1 [H + ] + K e1 K e2 [H + ] 2 + K es1 [H + ] +K es1 K es2 (6.18) 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 −1.0 −0.5 0.0 0.5 1.0 K 1d K 2a K 2b K 2c K 2d K 2e K 1c K 1b K 1a K 1e pH ( a ) ( b ) First Derivative 0.0 0.5 1.0 1.5 2.0 0.0 0.1 0.2 0.3 0.4 0.5 −log 10 (K 1 /K 2 ) |pK predicted −pK actual | Figure 6.5. (a) Simulation of log V max /K s or log V max patterns as a function of the close- ness between K 1 and K 2 values in the enzyme. (b) Errors between actual and predicted pK values as a function of the difference in pK values of the catalytic groups in the enzyme. NEW METHOD OF DETERMINING pK VALUES OF CATALYTIC GROUPS 87 2 3 4 5 6 7 8 9 10 −1.0 −0.5 0.0 0.5 1.0 pK e1 =5.8 pK e2 =6.9 pK e1 =5.7 pK e2 =6.8 pH ( a ) ( b ) ∆log( V * max /K s * )/∆pH 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 y + =0.99x−0.66 y − =−0.88x+11 y 0 =5 pH log 10 (V * max /K s * ) Figure 6.6. (a) pH dependence of the slope of a log V max /K s versus pH data set. (b)pH dependence of a log V max /K s versus pH data set. A logarithmic transformation of Eq. (5.18), results in the expression −log K ∗ s =−log K s K es1 K e1 − log([H + ] 2 + K e1 [H + ] +K e1 K e2 ) + log([H + ] 2 + K es1 [H + ] +K es1 K es2 (6.19) The first derivative of Eq. (6.19) as a function of −log[H + ] (i.e., pH) is d(−log K ∗ s ) d(pH) = 2[H + ] 2 + K e1 [H + ] [H + ] 2 + K e1 [H + ] +K e1 K e2 − 2[H + ] 2 + K es1 [H + ] [H + ] 2 + K es1 [H + ] +K es1 K es2 (6.20) It is not as easy to calculate a value for this derivative at [H + ] = K,since the exact value will depend not only on the relative magnitude of K e1 88 pH DEPENDENCE OF ENZYME-CATALYZED REACTIONS 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 −1.0 −0.5 0.0 0.5 1.0 1.5 K es K e −logK s * logV * max /K s * logV * max /K s * logV * max logV * max pH ( a ) 234567 0.00 0.25 0.50 0.75 1.00 pK es pK e −logK s * pH ( b ) Slope Figure 6.7. (a) Simulation of the pH dependence of the logarithm of the catalytic param- eters V max , V max /K s ,andK s for a monoprotic enzyme. (b) Variation in the slope of the log V max ,logV max /K s ,and−logK s versus pH plots as a function of pH for a monopro- tic enzyme. versus K e2 , but also of K es1 versus K es2 . We do not recommend working with this expression, since the results obtained can be ambiguous. Caution must be exercised when using this approach to determine the pK values of the catalytic groups since considerable error can be introduced in their determination if they happen to be numerically close. Figure 6.5(a) is a simulation of log 10 (V ∗ max /K ∗ s ) or log 10 V ∗ max versus pH patterns as a function of the closeness between K 1 and K 2 values. Figure 6.5(b) shows the error between actual and predicted pK values as a function of the difference between pK values. Our simulation shows NEW METHOD OF DETERMINING pK VALUES OF CATALYTIC GROUPS 89 that as long at the difference between pK values is greater than 1 pH unit, the error introduced in the determination of pK values will be less than 0.1 pH unit. Figure 6.6(a) shows an actual analysis of the pH dependence of V ∗ max / K ∗ s for the hydration of fumarate by the enzyme fumarase. The slope of the line at the midpoint between two subsequent pH values was calculated from the data as slope (pH 2 −pH 1 )/2 = log Y ∗ 2 − log Y ∗ 1 pH 2 − pH 1 (6.21) where Y ∗ could correspond to V ∗ max /K ∗ s or V ∗ max . A general trend line through the data points was obtained by interpolation. From this trend line, the pH values at which the slope was +0.5 and −0.5 were easily determined. This procedure proved to be rapid, accurate, and reliable. In our experience, drawing straight lines through the usual small num- ber of data points, as carried out in the Dixon analysis, was not easy, particularly for the slope = 0 line. This ambiguity made it difficult to have confidence in the pK values determined. The procedure developed in this chapter is more reliable. On the other hand, the pK values obtained using the Dixon analysis and the analysis presented in this chapter were found to be similar (Fig. 6.6b). Before leaving this topic, we would like to draw to the attention of the reader that many enzymes may have only one ionizable group among their catalytic groups. For this case, the patterns obtained for the pH dependence of the catalytic parameters will be half that of their two-ionizable-group counterparts (Fig. 6.7). For this case, the determination of pK e and pK es values is less prone to error since there is no interference from a second ionizable group. CHAPTER 7 TWO-SUBSTRATE REACTIONS Up to this point, the kinetic treatment of enzyme-catalyzed reactions has dealt only with single-substrate reactions. Many enzymes of biological importance, however, catalyze reactions between two or more substrates. Using the imaginative nomenclature of Cleland, two-substrate reactions can be classified as ping-pong or sequential. In ping-pong mechanisms, one or more products must be released before all substrates can react. In sequential mechanisms, all substrates must combine with the enzyme before the reaction can take place. Furthermore, sequential mechanisms can be ordered or Random. In ordered sequential mechanisms, substrates react with enzyme, and products are released, in a specific order. In ran- dom sequential mechanisms, on the other hand, the order of substrate combination and product release is not obligatory. These reactions can be classified even further according to the molecularity of the kinetically important steps in the reaction. Thus, these steps can be uni (unimolecu- lar), bi (bimolecular), ter (termolecular), quad (quadmolecular), pent (pen- tamolecular), hexa (hexamolecular), and so on. This molecularity applies both to substrates and products. Using Cleland’s schematics, examples of ping-pong bi bi, ordered-sequential bi bi, and random-sequential bi bi reactions are, respectively, ABP (EA E′P) E′ (E′B EQ) Q EE (7.1) 90 RANDOM-SEQUENTIAL Bi Bi MECHANISM 91 AB P EA (EAB EPQ) Q EE EQ (7.2) BA Q P EEAB EPQ E BAQP (7.3) Irrespective of the mechanism, all two-substrate enzyme-catalyzed reac- tions of the type A + B ←−− −−→ P +Q (7.4) obey the equation v V  max = [S] K  + [S] (7.5) under conditions where the concentration of one of the two substrates is held constant while the other is varied. For Eq. (7.5), [S] corresponds to the variable substrate’s concentration, while K  and V  max correspond to the apparent Michaelis constant and apparent maximum velocity for the enzymatic reaction, respectively. Kinetic analysis of multiple substrate reactions could stop at this point. However, if more in-depth knowledge of the mechanism of a particular multisubstrate reaction is required, a more intricate kinetic analysis has to be carried out. There are a number of common reaction pathways through which two-substrate reactions can proceed, and the three major types are discussed in turn. 7.1 RANDOM-SEQUENTIAL Bi Bi MECHANISM For the random-sequential bi bi mechanism, there is no particular order in the sequential binding of substrates A or B to the enzyme to form the ternary complex EAB. A general scheme for this type of reactions is 92 TWO-SUBSTRATE REACTIONS E +A K A s −− −− EA ++ BB  | | | |  K B s  | | | |  K AB EB +A −− −− K BA EAB −−→ k cat E + P + Q (7.6) In this model we assume that rapid equilibrium binding of either substrate A or B to the enzyme takes place. For the second stage of the reaction, equilibrium binding of A to EB and B to EA, or a steady state in the concentration of the EAB ternary complex, may be assumed. The rate equation for the formation of product, the equilibrium dissocia- tion constant for the binary enzyme–substrate complexes EA and EB (K A s and K B s ), the equilibrium dissociation (K s ) or steady-state Michaelis (K m ) constants for the formation of the ternary enzyme–substrate complexes EAB (K AB and K BA ), and the enzyme mass balance are, respectively, v = k cat [EAB] (7.7) K A s = [E][A] [EA] K B s = [E][B] [EB] (7.8) K BA = [EB][A] [EBA] K AB = [EA][B] [EAB] [E T ] = [E] + [EA] + [EB] + [EAB] (7.9) A useful relationship exists among these constants: K A s K B s = K BA K AB (7.10) Normalization of the rate equation by total enzyme concentration (v/[E T ]) and rearrangement in light of Eq. (7.10) results in the rate equation for random-order bi bi mechanisms: v V max = [A][B] K A s K AB + K AB [A] +K BA [B] + [A][B] (7.11) where V max = k cat [E T ]. RANDOM-SEQUENTIAL Bi Bi MECHANISM 93 7.1.1 Constant [A] For the case where the concentration of substrate A is held constant, Eq. (7.11) can be expressed as v V  max = [B] K  + [B] (7.12) where V  max = V max [A] K BA + [A] (7.13) and K  = K A s K AB + K AB [A] K BA + [A] = K AB (K A s + [A]) K BA + [A] (7.14) From determinations of K  and V  max at different fixed concentrations of substrate A, it is possible to obtain estimates of V max , K A s , K AB ,and K BA . V  max displays a hyperbolic dependence on substrate A concentra- tion (Fig. 7.1a). Thus, by fitting Eq. (7.13) to V  max –[A] experimental data using nonlinear regression, it is possible to obtain estimates of V max and K BA . K  also displays a hyperbolic dependence on substrate A concentra- tion (Fig. 7.1b). However, this hyperbola does not go through the origin. At [A] = 0(y-intercept), K  = K A s K AB /K BA (or K B s ), while in the limit where [A] approaches infinity, K  = K AB (Fig. 7.1b). Thus, by fitting Eq. (7.14) to K  − [A] experimental data using nonlinear regression, it is possible to obtain estimates of K A s K AB /K BA and K AB . Since the values of K AB , K BA ,andK B s (y-intercept) are known, it is straightforward to obtainanestimateofK A s using Eq. (7.10): K A s = K B s K BA K AB (7.15) 7.1.2 Constant [B] For the case where the concentration of substrate A is held constant, Eq. (7.11) can be expressed as v V  max = [A] K  + [A] (7.16) where V  max = V max [B] K AB + [B] (7.17) 94 TWO-SUBSTRATE REACTIONS K BA K BA K AB K AB [A] ( a ) K s B K s A [A] ( b ) ( c )( d ) K′ [B] V′ max V′ max V max V max [B] K′ Figure 7.1. Fixed substrate concentration dependence for enzymes displaying random- sequential mechanisms: (a) Dependence of V  max on [A]; (b) dependence of K  on [A]; (c) dependence of V  max on [B]; (d) dependence of K  on [B]. and K  = K A s K AB + K BA [B] K AB + [B] = K B s K BA + K BA [B] K AB + [B] = K BA (K B s + [B]) K AB + [B] (7.18) From determinations of K  and V  max at different fixed concentrations of substrate B, it is possible to obtain estimates of V max , K B s , K AB ,and K BA . V  max displays a hyperbolic dependence on substrate B concentra- tion (Fig. 7.1c). Thus, by fitting Eq. (7.17) to V  max –[B] experimental data using nonlinear regression, it is possible to obtain estimates of V max and K AB . K  also displays a hyperbolic dependence on substrate B concentra- tion (Fig. 7.1d). However, this hyperbola does not go through the origin. At [B] = 0(y-intercept), K  = K B s K BA /K AB (or K A s ), while in the limit where [B] approaches infinity, K  = K BA (Fig. 7.1d). Thus, by fitting Eq. (7.18) to K  –[B] experimental data using nonlinear regression, it is possible to obtain estimates of K A s and K BA . Since the values of K AB , [...]... [A] (7.41) Vmax [A] A αKm + [A] (7.42) = B Km [A] A αKm + [A] (7.43) From determinations of K and Vmax at different fixed concentrations of A B substrate A, it is possible to obtain estimates of Vmax , αKm , and Km Vmax displays a hyperbolic dependence on substrate A concentration (Fig 7.3c) 100 TWO-SUBSTRATE REACTIONS A aKm K′ V′ max Vmax B Km B Km [B] [B] (a ) (b ) B Km K′ V′ max Vmax A aKm A aKm... case, n becomes merely an index of cooperativity, which can have noninteger values Estimates of the parameters k and n are obtained using standard nonlinear regression procedures available in most modern graphical software packages By fitting the Hill equation to experimental v versus [S] data, estimates of k , n, and Vmax can easily be obtained Simulations of v versus [S] behavior using Eq (8. 15) are... popular, the use of steady-state kinetic analysis to determine the mechanism of an enzymatic reaction has decreased in favor of pre-steadystate analysis of kinetic data obtained from rapid-reaction techniques CHAPTER 8 MULTISITE AND COOPERATIVE ENZYMES Many enzymes are oligomers composed of distinct subunits Often, the subunits are identical, each bearing an equivalent catalytic site If the sites are... the fashion described above The Hill constant, k , Hill coefficient, n, and Vmax are parameters used to characterize the catalytic properties of cooperative enzymes The Hill constant is related to the enzyme substrate dissociation constants (k = kn ) and provides an estimate of the affinity of the enzyme for a particular substrate The relationship between the Hill constant and the substrate concentration... EAB − → E + P + Q − (7.20) The rate equation for the formation of product, the equilibrium dissociA ation constant for the binary enzyme substrate complex EA (Ks ), the equilibrium dissociation (Ks ), or steady-state Michaelis (Km ) constant for the formation of the ternary enzyme substrate complex EAB (K AB ), and the enzyme mass balance are, respectively, v = kcat [EAB] [E] [A] [EA][B] A Ks = K AB... expressed as v Vmax = [A] K + [A] (7. 25) 96 TWO-SUBSTRATE REACTIONS where Vmax = and K = Vmax [B] K AB + [B] A Ks K AB K AB + [B] (7.26) (7.27) From determinations of K and Vmax at different fixed concentrations of A substrate B, it is possible to obtain estimates of Vmax , Ks , and K AB Vmax displays a hyperbolic dependence on substrate B concentration (Fig 7. 2a) Thus, by fitting Eq (7.26) to Vmax –[ B]... substrate concentration curve for a cooperative enzyme Enzyme activity can also be affected by binding of substrate and nonsubstrate ligands, which can act as activators or inhibitors, at a site other than the active site These enzymes are called allosteric These responses can be homotropic or heterotropic Homotropic responses refer to the allosteric modulation of enzyme activity strictly by substrate... is quite applicable in this instance It is difficult to determine the mechanism of an enzymecatalyzed reaction from steady-state kinetic analysis The determination of the mechanism of an enzymatic reaction is neither a trivial task nor an easy task The use of dead-end inhibitors and alternative substrates, study of the patterns of product inhibition, and isotope-exchange experiments DIFFERENTIATION BETWEEN... [EA] [EAB] [ET ] = [E] + [EA] + [EAB] (7.21) (7.22) (7.23) Normalization of the rate equation by total enzyme concentration (v/[ET ]) and rearrangement results in the rate equation for ordered-sequential bi bi mechanisms: v Vmax = A Ks K AB [A] [B] + K AB [A] + [A] [B] (7.24) where Vmax = kcat [ET ] 7.2.1 Constant [B] For the case where the concentration of substrate B is held constant, Eq (7.24) can... substrate A concentration (Fig 7.2c) The slope of A this function equals Ks K AB In the limit where [A] approaches infinity, K = K AB (Fig 7.2c) Thus, by fitting Eq (7.30) to K – [A] experimental A data using nonlinear regression, it is possible to obtain estimates of Ks AB and K ORDERED-SEQUENTIAL Bi Bi MECHANISM 97 V′ max Vmax KAB [B] (a ) K′ A Ks [B] K′ (b ) KAB [A] (c ) Figure 7.2 Fixed substrate . ternary enzyme substrate complexes EAB (K AB and K BA ), and the enzyme mass balance are, respectively, v = k cat [EAB] (7.7) K A s = [E] [A] [EA] K B s = [E][B] [EB] (7.8) K BA = [EB] [A] [EBA] K AB = [EA][B] [EAB] [E T ]. [B] (7.12) where V  max = V max [A] K BA + [A] (7.13) and K  = K A s K AB + K AB [A] K BA + [A] = K AB (K A s + [A] ) K BA + [A] (7.14) From determinations of K  and V  max at different fixed concentrations. enzyme substrate complex EAB (K AB ), and the enzyme mass balance are, respectively, v = k cat [EAB] (7.21) K A s = [E] [A] [EA] K AB = [EA][B] [EAB] (7.22) [E T ] = [E] + [EA] + [EAB] (7.23) Normalization