title: The role of glycine 99 in L-lactate monooxygenase from Mycobacterium smegmatis.
abstract: Glycine 99 in L-lactate monooxygenase (LMO) from Mycobacterium smegmatis was mutated to serine and threonine, and the resultant mutants were studied extensively to explore the role of this residue in maintaining monooxygenase activity and in controlling the reactivity with molecular oxygen. Both mutants were observed to lose monooxygenase activity completely and generate H2O2 and pyruvate as reaction products. However, the mutants have much lower activities than a true L-lactate oxidase. The oxygen reactivities of the reduced and semiquinone forms of the mutant enzymes were significantly different from those of wild type enzyme. These results confirm our previous suggestion that the electronic interactions in the active site are a crucial factor that governs the 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 lower stabilization of the transition state.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
authors.forename: W
authors.initials: W
authors.lastname: Sun
authors.forename: C H
authors.initials: CH
authors.lastname: Williams
authors.forename: V
authors.initials: V
authors.lastname: Massey
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D005998:Glycine; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009161:Mycobacterium / Q000201:enzymology*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013237:Stereoisomerism; D013379:Substrate Specificity; D013447:Sulfites / Q000378:metabolism
chemical_list: D007773:Lactates; D011994:Recombinant Proteins; D013447:Sulfites; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase; D005998:Glycine