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A mutant form of L-lactate oxidase (LOX) from Aerococcus viridans in which 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 chain ␣-hydroxyacid oxidase from rat kidney. A95G-LOX keeps oxidase activity with L-lactate at the same level as wild type LOX but has much enhanced oxidase activity with longer chain L-␣-hydroxyacids, ␣-hydroxy-n-butyric acid, ␣-hydroxy-n-valeric acid, etc., and also the aromatic ␣-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 A95G-LOX. The affinity of pyruvate for the reduced enzyme is increased, and sulfite binding to the oxidized enzyme is weaker in A95G-LOX than in 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.␣-Hydroxyacid oxidizing enzymes, a family of flavoprotein enzymes, share a series of common characteristics. Within the family, the crystal structures of glycolate oxidase and flavocytochrome b 2 have been solved by x-ray diffraction studies (1-4). The reaction mechanisms of L-lactate monooxygenase from Mycobacterium smegmatis and flavocytochrome b 2 from Saccharomyces cerevisiae have been studied extensively (5, 6). L-Lactate monooxygenase utilizes L-lactate and other L-␣-hydroxyacids but is unique in accelerating the oxidative decarboxylation of the products, pyruvate (or corresponding keto acid) and hydrogen peroxide, to acetic acid (or corresponding carboxylic acid), carbon dioxide, and water. New members of this flavoenzyme family have been described including those utilizing aromatic and bulky substrates like mandelate and long chain ␣-hydroxyacids (7-9).We are studying one of the new enzymes of this family, L-lactate oxidase from Aerococcus viridans (10). This enzyme utilizes L-lactate and oxygen as L-lactate monooxygenase does but forms pyruvate and hydrogen peroxide as the final products instead of catalyzing the oxidative decarboxylation reaction. We believe that lactate oxidase is an ideal enzyme for studying the reaction mechanism of this enzyme family because of its considerable stability, and it is also a useful enzyme for the construction of a lactate sensor for biological applications.By analogy with the crystal structures of glycolate oxidase and flavocytochrome b 2 , it has been pointed out that glycine 99 in L-lactate monooxygenase is unique in the family of enzymes (except now mandelate dehydrogenase (11) also with glycine), because the other family members have an alanine residue at the homologous position (6). This residue, in glycolate oxidase and flavocytochrome b 2 , is in close contact with the flavin N-5 position on the re-face of the flavin. It was considered possible that the s...

ilvE gene of Escherichia coli was inserted into the region downstream of the tac promotor. As a result, the branched-chain amino acid aminotransferase was overproduced by about a hundred-fold in E. coli W3110. The overproduced aminotransferase was purified from cell extracts about 40-fold to homogeneity. Chemical and physicochemical analyses confirmed that it was a product of the ilvE gene. The enzyme existed in a hexamer with a subunit molecular weight of 34,000; the double trimer model of the enzyme presumed by the previous chemical cross-linking experiments (Lee-Peng, F.-C. et al. (1979) J. bacteriol. 139, 339-345) was supported by electron micrographs. The circular dichroic (CD) spectrum of branch-chain amino acid aminotransferase had double negative maxima at 210 and 220 nm. The alpha-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. The absorption spectrum of the enzyme showed two peaks at 330 and 410 nm. There was no pH-dependent spectral shift. The CD spectrum of the coenzyme, pyridoxal 5'-phosphate, had negative peaks at 330 and 410 nm. These spectral properties of branched-chain amino acid aminotransferase were quite different from those of E. coli aspartate aminotransferase. Each subunit bound approximately 1 mol of pyridoxal 5'-phosphate. A lysyl residue, which forms a Schiff base with the aldehyde group of the pyridoxal 5'-phosphate, was identified in the primary structure of the enzyme.

The rate constants for reduction of the f lavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1 H-and 2-2 H-forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects ( 1 H͞ 2 H ‫؍‬ 3-7) on the rate constants of f lavin reduction were found, indicating that f lavin reduction is a direct measure of ␣-C-H bond breakage. The rate constants show only a small inf luence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the f lavin N(5) position or a synchronous mechanism in which the ␣-C-H is formally abstracted as a H ؉ while the resulting charge is simultaneously neutralized by another event.L-Lactate oxidase is a newly studied member of the family of FMN-containing enzymes that catalyze the oxidation of ␣-hydroxyacids, comprising glycolate oxidase (EC 1.1.3.1), Llactate oxidase, L-lactate monooxygenase (EC 1.13.12.4), flavocytochrome b 2 (EC 1.1.2.3), long-chain ␣-hydroxyacid oxidase (EC 1.1.3.15), and L-mandelate dehydrogenase. A considerable amount of mechanistic work has been carried out with L-lactate monooxygenase and with flavocytochrome b 2 (for reviews, see refs. 1 and 2), which has been interpreted as supporting a carbanion mechanism, in which the substrate ␣-proton is abstracted by an active site base and the electrons from the resulting substrate carbanion are transferred to the flavin. The crystal structures of two of the family members, glycolate oxidase (3) and flavocytochrome b 2 (4), have been solved and show an arrangement of conserved protein residues surrounding the FMN prosthetic group and substrate that are consistent with such a carbanion mechanism. In these structures, the substrate is positioned on the si-face of the flavin with the carboxylate in ionic interaction with arginine residues and stabilized by H-bond interaction with a tyrosine residue (5). A conserved histidine residue is located so that the ␣-carbon of the substrate would be positioned between the flavin N(5) and the histidine N1. The histidine residue therefore has been envisaged as the active site base responsible for abstracting the proton from the ␣-position of the substrate, with concomitant attack of the highly nucleophilic carbanion on flavin N(5).While the crystal structures of only two of the family members are available so far, the amino acid sequences of all the enzymes are known: glycolate oxidase (6, 7), flavocytochrome b 2 (8), lactate monooxygenase (9), lactate oxidase (10), long chain ␣-hydroxyacid oxidase (11), and mandelate dehydrogenase (12, 13). All enzymes of the group show considerable homology, and all have the strictly conserved set o...