SITE-DIRECTED MUTAGENESIS OF GLYCINE-99 TO ALANINE IN L-LACTATE MONOOXYGENASE FROM MYCOBACTERIUM-SMEGMATIS

L-Lactate monooxygenase (LMO) hom Mycobacterium smegmatis was mutated at glycine 99 to alanine, and the properties of the resulting mutant ( referred to as G99A) were studied, Mutant G99A of LMO was designed to test the postulate that the smaller glycine residue in the vicinity of the alpha-carbon methyl group of lactate in wild-type LMO has less st eric hindrance, leading to the retention and oxidative decarboxylation of pyruvate in the active site, a unique property of LMO in contrast to other members of the FMN-dependent oxidase/dehydrogenase family, G9 9A has been shown to be readily reduced by L-lactate at a rate similar to that of the wild-type enzyme. The binding of pyruvate to reduced G 99A is 4-fold weaker than that to the wild-type enzyme. A dramatic cha nge of this mutation is that G99A has a much lower oxygen reactivity t han the wild-type enzyme. Pyruvate-bound reduced G99A reacts with O-2 at a rate similar to 10(5)-fold slower than the wild-type enzyme, and free reduced G99A reacts with O-2 at a rate similar to 100-fold slower than the wild-type enzyme. Due to the very low oxygen reactivity of t he pyruvate-bound reduced enzyme, G99A has been shown to catalyze the oxidation of L-lactate to pyruvate and hydrogen peroxide instead of ac etate, carbon dioxide, and water, the normal decarboxylation products of pyruvate and hydrogen peroxide. Thus, the mutation alters the enzym e from its L-lactate monooxygenase activity to L-lactate oxidase activ ity, However, compared with L-lactate oxidase, G99A has a much lower r eactivity toward oxygen Our results also reveal that the small steric change around N-5 of the flavin causes a profound change in the electr onic distribution in the catalytic cavity of the enzyme and imply that electrostatic interactions in the active site provide an important fa ctor for control of O-2 reactivity.