What limits the rate of an enzyme-catalyzed reaction

Abstract

In recent years there has been some controversy about how enzymes manage to catalyze chemical reactions as fast as they do. Lost sight of in these arguments, however, has been the fact that the actual chemical reaction is seldom what limits the rate of an enzymatic reaction.It is the purpose of this Account to show that conformation changes in the protein and release of products (or more likely, the conformation change that permits product release) are the common rate-limiting steps. We present several well-documented cases and the kinetic evidence that allows us to deduce the relative rates of the various steps in the mechanism. Finally we show that the real upper limit on the rate of an enzyme-catalyzed reaction of a thermodynamic one.Basic Theory. When the concentration of one substrate is varied and the levels of others held constant, most enzyme-catalyzed reactions obey the rate law where [A] is substrate concentration, V the maximum obtainable velocity at infinite substrate concentration, and K the Michaelis constant, which is the level of substrate at which the velocity is half of V and is thus the apparent dissociation constant for the enzyme-substrate complex under steady-state conditions. When [A] is infinite, = V, but as [A] approaches zero, v = {V/K)[k}, so that V/K is the apparent first-order rate constant at low substrate concentration. V and V/K are the two fundamental constants that determine the kinetics, and they vary independently with the concentrations of other substrates, products, inhibitors, or activators, or with pH, temperature, ionic strength, etc. The variation of K with all these factors is simply determined by the ratio of Vand V/K.

Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

“…10 Indeed, in recent years much debate has emerged regarding the role of protein dynamics in enzyme catalysis in general 5,11,12 and in DHFR, in particular. 6,[13][14][15][16][17][18] How protein dynamics might contribute to catalysis ranges from conformational gating, where conformational changes might even be rate limiting, 19,20 as is thought to be the case with DHFR, [21][22][23] to a much more speculative direct coupling of specific protein motions to the reaction coordinate, perhaps similar to the strong and selective Fermi resonance between the stretching and bending motions of C-H moieties. [24][25][26] Unfortunately, the study of protein electrostatics and dynamics has been limited by the challenges associated with the direct characterization of specific protein bonds, including their environment and motion.…”

Limits to Catalysis by Ribonuclease A

“…The rate of UpA cleavage by RNase A is maximal at pH 6.0, where k cat = 1.4 × 10 3 s −1 and k cat /K m = 2.3 × 10 6 M −1 s −1 at 25°C. At pH 6.0 and 25°C, the uncatalyzed rate of [5, H]Up [3,5, H]A cleavage was found to be k uncat = 5 × 10 −9 s −1 (t 1/2 = 4 years). Thus, RNase A enhances the rate of UpA cleavage by 3 × 10 11 -fold by binding to the transition state for P-O 5′ bond cleavage with a dissociation constant of <2 × 10 −15 M.…”

Genetic variation and relative catalytic efficiencies: lactate dehydrogenase B allozymes of Fundulus heteroclitus.

“…In the present study we are concerned with nonparametric estimates because they are more robust than least-squares estimates (21 [2] There are two fundamental rate constants in Eq. 2 that determine the overall kinetics (25): kcat (units, s'l), the limiting velocity observed as the pyruvate molarity tends toward infinity, and the apparent second-order rate constant kcat/Km (units, M-'s-'). As pyruvate concentrations are lowered, the reaction velocity (v) approaches first order relative to the pyruvate molarity, and Eq.…”