CONVERSION OF L-LACTATE OXIDASE TO A LONG-CHAIN ALPHA-HYDROXYACID OXIDASE BY SITE-DIRECTED MUTAGENESIS OF ALANINE-95 TO GLYCINE

A mutant form of L-lactate oxidase (LOX) from Aerococcus viridans in w hich alanine 95 was replaced by glycine was constructed as a mimic of L-lactate monooxygenase but proved instead to be a mimic of the long c hain alpha-hydroxyacid oxidase from rat kidney. A95G-LOX keeps oxidase activity with L-lactate at the same level as wild type LOX but has mu ch enhanced oxidase activity with longer chain L-alpha-hydroxyacids, a lpha-hydroxy-n-butyric acid, alpha-hydroxy-n-valeric acid, etc., and a lso the aromatic alpha-hydroxyacid, L-mandelic acid. Kinetic analysis of the activity with these substrates indicates that the reduction of the enzyme bound flavin by substrates is the rate-limiting step in A95 G-LOX. The affinity of pyruvate for the reduced enzyme is increased, a nd sulfite binding to the oxidized enzyme is weaker in A95G-LOX than i n native enzyme. Wild type LOX stabilizes both the neutral and anionic flavin semiquinones with a pK(a) of 6.1, but A95G LOX stabilizes only the anionic semiquinone form. These results strongly suggest that the environment around the N5-C4a region of the flavin isoalloxazine ring is changed by this mutation.