Energetics of proline racemase: tracer perturbation experiments using [14C]proline that measure the interconversion rate of the two forms of free enzyme

Abstract

Proline racemase exists in two states, one of which binds the isomerizes L-proline and the other of which binds and isomerizes D-proline. In the enzyme-catalyzed racemization of proline at high substrate concentrations, the interconversion of the two forms of the free enzyme becomes rate limiting. The tracer perturbation method of Britton (1966, 1973) vividly demonstrates the kinetic importance of this enzyme interconversion under oversaturating conditions and allows an estimate of the rate constant for this reaction of 105 s-1. It is further shown that the enzyme is bound state saturated and that the peak-switch concentration, cp, is 125 mM. At substrate concentrations higher than 125 mM the enzyme becomes oversaturated, and the reaction rate is limited by the transition state for the interconversion of the two forms of the free enzyme. It seems likely that the two free enzyme forms differ only in the protonation states of the acidic and basic groups at the active site.