ASSAY AND INHIBITION OF GLYOXALASE I

Glyoxalase I is an enzyme which catalyzes the conversion of hemimercaptals derived from substituted glyoxals and glutathione to the corresponding substituted thioesters of (alpha)-hydroxyacetic acid. This reaction is thought to proceed through an enediol intermediate. Inhibition of this enzyme was investigated in order to delineate the steric and mechanistic factors which are important to its mechanism of action. A variety of S-substituted glutathione derivatives were synthesized and found to be good to excellent competitive inhibitors of glyoxalase I. Ki's ranged from 10('-3) M to < 10('-5) M for a series of S-alkyl-, S-carbamoyl-, S-acyl-, and S-(acylmethyl)-glutathiones. New compounds prepared and evaluated included S-(n-butylcarbamoyl)-, S-(cyclohexylcarbamoyl)-, S-(5-iodopentylcarbamoyl)-, and S-(ethoxycarbonylcarbamoyl)-glutathiones, S-phenylacetyl glutathione, S-trans-cinnamyl glutathione, S-trans-cinnamoyl glutathione, and S-(p-phenylphenacyl)-glutathione. No inhibition was observed for any compound in a group which included hydroxamic acids, (beta)-keto carboxylic acid derivatives, and small molecules with enediol structures. Some of the compounds investigated absorb strongly at 240 nm, the wavelength of observation for the usual assay employing methylglyoxal as the enzyme cosubstrate. It was demonstrated that compounds which themselves absorb strongly at the wavelength of measurement may give an apparent decrease in the absorbance changes on which enzyme activity calculations are based, even when these compounds are contained in separate absorption cells. An alternative assay of glyoxalase I activity employing phenylglyoxal, a stable crystalline solid, as substrate was developed. The method, which is based on observation of the decrease in absorbance at 263 nm, the phenylglyoxal-glutathione hemimercaptal isosbestic point, is reliable, precise, and convenient. It proved useful both for routine assays and for evaluation of strongly-absorbing compounds for which the standard methylglyoxal assay gave inaccurate results. It was concluded that inhibition is optimized when one end of the inhibitor is glutathione and the other end is bulky and hydrophobic, preferably containing one or more aromatic rings. The exact structure of the middle region is much less important, even when it either is an analog of the presumed enediol intermediate or can enolize to such a structure.