THE ROLE OF GLYCINE-99 IN L-LACTATE MONOOXYGENASE FROM MYCOBACTERIUM-SMEGMATIS

Glycine 99 in L-lactate monooxygenase (LMO) from Mycobacterium smegmat is was mutated to serine and threonine, and the resultant mutants were studied extensively to explore the role of this residue in maintainin g monooxygenase activity and in controlling the reactivity with molecu lar oxygen. Both mutants were observed to lose monooxygenase activity completely and generate H2O2 and pyruvate as reaction products. Howeve r, the mutants have much lower activities than a true L-lactate oxidas e. The oxygen reactivities of the reduced and semiquinone forms of the mutant enzymes were significantly different from those of wild type e nzyme. These results confirm our previous suggestion that the electron ic interactions in the active site are a crucial factor that governs t he oxygen reactivity of the enzyme (Sun, W., Williams, C. H., Jr., and Massey, V. (1996) J. Biol. Chem. 271, 17226-17233). In addition, the mutants cause a dramatic decrease of the rate of flavin reduction by L -lactate compared with the wild type enzyme, mainly due to the much lo wer stabilization of the transition state.