Studies on the mechanism of action of the flavoenzyme lactate oxidase : proton uptake and release during the binding of transition state analogs

Abstract

Oxalate and malonate have been found to bind to the flavoenzyme, lactate oxidase, in a two-step equilibrium process. The dissociation constant for the first step is of the order of lo power -2 M, and is independent of pH. The second step equilibrium is on the other hand directly proportional to [H+] due to the second forward step, which is a second order reaction involving proton uptake. The reverse of this step is a first order reaction involving proton release, and consequently is independent of pH. Simultaneous measurement of proton uptake and formation of the enzyme. oxalate complex show that the rate of proton uptake is coincident with the large spectral changes occurring on binding, with the stoichiometry of one proton uptake per equivalent of enzyme-bound flavin. These results give strong support to the concept that oxalate and malonate are transition state analogs of a carbanion form of the substrate, formed in an early step in the enzyme reaction mechanism. The enzyme base responsible for abstraction of the proton from the (α-carbon of the substrate is postulated to be the same one which is protonated (from the medium) in the second step of binding of oxalate and malonate, to form the transition state equivalent to that occurring with the substrate carbanion in catalysis. The pK of this group has been determined as 4.7, and is shifted to 8.2 and 9.8 on binding with malonate and oxalate, respectively. These pK shifts represent stabilizations of ~5 and ~7 kcal/mole of the enzyme inhibitor complexes. These findings offer strong support for the intermediate involvement of a substrate carbanion in catalysis.