Lactate monooxygenase. II. Site-directed mutagenesis of the postulated active site base histidine 290.
Lactate monooxygenase catalyzes the oxidation of L-lactate with molecular oxygen to acetate, CO2, and water. Histidine 290 has been proposed to be the active site base in lactate monooxygenase (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632) and was mutated to a glutamine (H290Q). The mutant enzyme shows properties that support strongly the postulated function of the histidine. The ability of L-lactate to reduce the enzyme flavin is essentially abolished, whereas reoxidation of reduced enzyme with oxygen proceeds at 1.4 x 10(4) M-1 s-1, a rate essentially like that found in the wild type enzyme. The substrate, L-lactate, is bound with a Kd equal to 2.0 x 10(-2) M, and D-lactate, a competitive inhibitor with a Kd of 3.1 x 10(-3) M. Both values are similar to binding measured in the wild type enzyme. Unlike the situation with wild type enzyme, where the transition state analog oxalate is bound tightly in a two-step reaction involving proton uptake from solution (Ghisla, S., and Massey, V. (1977) J. Biol. Chem. 252, 6729-6735), the mutant enzyme binds oxalate weakly, in a single step reaction, with a Kd in the order of 0.1 M. No effect was observed upon varying the pH, indicating that binding does not include a protonation step. Replacing the histidine also has a significant effect on the ability of the enzyme to stabilize the flavin N(5)-sulfite adduct. Sulfite is bound at least 1000-fold weaker than it is in the wild type enzyme.
Müh
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Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Williams
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Massey
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D006639:Histidine; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013447:Sulfites / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013447:Sulfites; D006639:Histidine; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase