Purification and characterization of 3-mercaptolactic acid S-conjugate oxidases.
Two enzymes catalysing the oxidative formation of 3-mercaptopyruvic acid S-conjugates from L-3-mercaptolactic acid S-conjugates were purified to apparent homogeneity from rat liver cytosol. The two enzymes, tentatively designated MLO-I and MLO-II, showed a molecular mass of 160 and 250 kDa and were composed of four and six subunits of 41 and 39 kDa, respectively. Both enzymes possessed flavin mononucleotide as prosthetic group and oxidized several aromatic and aliphatic S-substituted L-3-mercaptolactic acids as well as alpha-hydroxy acids such as L-3-phenyllactic acid and L-2-hydroxyisocaproic acid. Glycolic acid and 3-(4-hydroxyphenyl)-lactic acid were the specific substrates for MLO-I and MLO-II, respectively. Neither of the enzymes oxidized beta- and gamma-hydroxy acids such as 3- and 4-hydroxybutyric acid. 2-Hydroxyisobutyric acid, ethyl-2-hydroxybutyrate, malic acid, 1-butanol, benzyl alcohol and L-leucine did not act as substrates for the enzymes. MLO-I and MLO-II exerted their maximum activities around pH 5.5 with Km of 0.5 and 0.25 mM and Vmax of 0.9 and 0.2 mumol/min/mg, respectively, when S-(4-bromophenyl)-3-thiolactic acid was used as substrate. MLO-I was inhibited by sulphydryl-modifying agents, while MLO-II was not. Both enzymes were strongly inhibited by divalent metal ions. These results indicate that MLO-I and MLO-II are different from L-amino acid oxidase (EC 1.4.3.2), malate oxidase (EC 1.1.3.3), L-alpha-hydroxy acid oxidase (EC 1.1.3.15) and glycolate oxidase (EC 1.1.3.1). The present enzymes are likely to be involved in the formation of cysteine conjugates from L-3-mercaptolactic acid S-conjugates in conjunction with cysteine conjugate aminotransferases.
Tomisawa
H
H
Drug Metabolism and Analytical Chemistry Research, Upjohn Pharmaceuticals Limited Tsukuba Research Laboratories, Ibaraki, Japan.
Hayashi
M
M
Uda
F
F
Okamoto
A
A
Hattori
K
K
Ozawa
N
N
Tateishi
M
M
D000818:Animals; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology; D008401:Gas Chromatography-Mass Spectrometry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D013438:Sulfhydryl Compounds; C008361:beta-mercaptolactate; D010088:Oxidoreductases; D003545:Cysteine