Inactivation of yeast glutathione reductase by O-phthalaldehyde.
Yeast glutathione reductase was inactivated by the bifunctional reagent, o-phthalaldehyde. The initial rate of inactivation followed pseudo-first order kinetics. Fluorescence spectral properties of modified enzyme indicated the formation of an isoindole derivative from cysteine and lyaine residues present in close proximity as shown by typical fluorescence emission and excitation maximum at 410 nm and 337 nm, respectively. The fluorescence spectral studies with o-phthalaldehyde in the presence and absence of N-ethylmaleimide indicated that both the inhibitors react with the same cysteine residue, which is non-essential for enzyme activity. The coenzyme NADPH did not protect the enzyme against the o-phthalaldehyde reaction while oxidised glutathione prevented o-phthalaldehyde inactivation. This could be due to reaction of the amino group of glutathione with o-phthalaldehyde. Stoichiometry of the reaction showed that the formation of approximately 2 isoindole derivatives per subunit of glutathione reductase is accompanied by 75% loss of activity. The results suggest that o-phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation.
Pandey
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A
Department of Chemistry, Indian Institute of Technology, Kanpur, India.
Katiyar
S S
SS
D003545:Cysteine / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000187:drug effects; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D015003:Yeasts / Q000201:enzymology*; D009764:o-Phthalaldehyde / Q000494:pharmacology*
D013439:Sulfhydryl Reagents; D009764:o-Phthalaldehyde; D005980:Glutathione Reductase; D008239:Lysine; D003545:Cysteine; D005033:Ethylmaleimide