PubMed:8132520 JSONTXT 21 Projects

Lactate monooxygenase. III. Additive contributions of active site residues to catalytic efficiency and stabilization of an anionic transition state. Lactate monooxygenase catalyzes the conversion of L-lactate to acetate, CO2, and water with incorporation of molecular oxygen. Several amino acid residues of lactate monooxygenase had been postulated to interact in specific ways with the bound substrate (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632). Tyrosine 44 and arginine 293 were proposed to form a hydrogen bond and a salt bridge to the carboxyl-moiety of lactate. Tyrosine 152 was suggested to form a hydrogen bond to the alpha-hydroxyl group and could be involved in stabilizing a transient carbanionic intermediate of the substrate. The tyrosine residues were replaced with phenylalanines (Y44F, Y152F), and arginine 293 was mutated to a lysine (R293K). In all cases catalysis was significantly decreased; however, the binding affinity for L-lactate did not decrease. Instead, the Kd measured for Y152F was 10-fold lower than that for the wild type enzyme. The products of turnover with Y152F were similar to those with wild type enzyme, with 70-80% of the reaction proceeding to form acetate, CO2, and H2O. The catalytic reactions with both Y44F and R293K were substantially uncoupled, with between 60 and 80% of the catalytic turnover forming pyruvate and H2O2. For all mutant forms the reoxidation of enzyme with oxygen in the absence of pyruvate occurred at a rate similar to that measured for the wild type enzyme. The most important effect of the mutations was in the ability to stabilize the transition state analog oxalate. A linear relationship was found between the rate of reduction of the enzyme flavin and the dissociation constant for the binding of oxalate, demonstrating that many individual residues contribute to the lowering of the energy of the transition state, in addition to specific functions being assignable to some specific residues.