Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Abstract

Glyoxalase I from yeast (S. cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The MW of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approximately 32,000) than that of the enzyme from mammals (approximately 46,000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of 2 very similar or identical subunits. The enzymes contain 1 Zn atom/subunit. The isoelectric points (at 4.degree. C) for the yeast and mammalian enzymes are at pH 7.0 and 4.8, respectively; tryptic-peptide maps display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. However, the similarities demonstrated in mechanistic and kinetic properties indicate convergent evolution. The kcat. [molecular activity] and Km values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2 mM). Glyoxalase I from all sources investigated had a kcat./Km value near 107 .cntdot. s-1 .cntdot. M-1, which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behavior may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.