The roles of two amino acid residues in the active site of L-lactate monooxygenase - Mutation of arginine 187 to methionine and histidine 240 to glutamine

Lactate monooxygenase (LMO) catalyzes the conversion of L-lactate to acetat e, CO2, and water with the incorporation of molecular oxygen. Arginine 187 of LMO is highly conserved within the family of L-alpha-hydroxyacid oxidizi ng enzymes (LB, K. H. D., and Lederer, F. (1991) J. Biol. Chem. 266, 20877- 20881). By comparison with the equivalent residue in flavocytochrome b(2) f rom Saccharomyces cerevisiae (Pike, A. D., Chapman, S. K, Manson, F. D. C,, Reid, G. A, Gondry, M., and Lederer, F. (1996) in Flavins and Flavoprotein s (Stevenson, H. J., Massey, V., and Williams, C. Il., Jr., eds) pp. 571-57 4, University of Calgary Press, Calgary, AB, Canada), arginine 187 might be expected to have an important role in catalytic efficiency and substrate b inding in LMO. Histidine 240 is predicted to be close to the substrate bind ing site of LMO, although it is not conserved within the enzyme family. Arg inine 187 has been replaced with methionine (R187M), and histidine 240 has been replaced with glutamine(H240Q). L-Lactate oxidation by R187M is very slow. The binding of L-lactate to the mutant enzyme appears to be very weak, as is the binding of oxalate, a tran sition state analogue. The binding of pyruvate to the reduced enzyme is als o very weak, resulting in complete uncoupling of enzyme turnover, with H2O2 and pyruvate as the final products. In addition, anionic forms of the flav in are unstable. The K-d for sulfite is increased nearly 400-fold by this m utation. The semiquinone form of R187M is also thermodynamically unstable, although the overall midpoint potential for the two-electron reduction of R 187M is only 34 mV lower than for the wild-type enzyme. H240Q more closely resembles the wild-type enzyme. The steady-state activity of H240Q is compl etely coupled. The k(cat) is similar to that for the wild-type enzyme.