PURIFICATION, CHARACTERIZATION AND KINETIC STUDIES OF GLYOXALASE-I FROM RAT-LIVER

Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from rat liver has been purified about 5000-fold to an electrophoretically pure form with a specific activity of 950 units/mg. One of the purification steps was affinity chromatography on S-hexylglutathione bound to Sepharose 4B. The molecular weight of the enzyme was 54 000 as estimated by gel filtration. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave a subunit molecular weight of 27 000. Peptide mapping of a tryptic digest of the enzyme showed that the two subunits are very similar or identical. The isoelectric point of the enzyme was at pH 4.7 (at 4°C). The protein molecule (dimer) contained 2 sulfhydryl groups and the additional 24 half-cystines found by amino acid analysis were ascribed to 12 disulfide bonds. The enzyme was inactivated by amino-group reagents, but appeared not to be dependent on sulfhydryl groups for catalytic activity. Purified glyoxalase I was found to contain 2 g-atoms of Zn/mol (dimer). The apoenzyme was catalytically inactive, but activity was partially restored by Zn2+ and some other bivalent metal ions. The steady-state kinetics were studied with both methylglyoxal and phenylglyoxal as α-ketoaldehyde substrates. Glutathione appeared to act as a nonlinear competitive inhibitor versus its hemimercaptal adducts of the α-ketoaldehydes. Simulation studies demonstrated that the non-hyperbolic kinetics are not caused by bias introduced by possible errors in the determination of substrate concentrations, but are true reflexions of the rate behaviour of the enzyme. © 1979.