INACTIVATION OF YEAST GLUTATHIONE-REDUCTASE BY O-PHTHALALDEHYDE

Yeast glutathione reductase was inactivated by the bifunctional reagen t, o-phthalaldehyde. The initial rate of inactivation followed pseudo- first order kinetics, Fluorescence spectral properties of modified enz yme indicated the formation of an isoindole derivative from cysteine a nd lysine residues present in close proximity as shown by typical fluo rescence emmision and excitation maximum at 410 nm and 337 nm, respect ively. The fluorescence spectral studies with o-phthalaldehyde in the presence and absence of N-ethylmaleimide indicated that both the inhib itors react with the same cysteine residue, which is non-essential for enzyme activity. The coenzyme NADPH did not protect the enzyme agains t the o-phthalaldehyde reaction while oxidised glutathione prevented o -phthalaldehyde inactivation. This could be due to reaction of the ami no group of glutathione with o-phthalaldehyde. Stoichiometry of the re action showed that the formation of approximately 2 isoindole derivati ves per subunit of glutathione reductase is accompanied by 75% loss of activity. The results suggest that o-phthalaldehyde binds to non-esse ntial cysteine and lysine residues present in close proximity which re sults in conformational changes leading to enzyme inactivation.