IDENTIFICATION OF CYSTEINE AND LYSINE RESIDUES PRESENT AT THE ACTIVE-SITE OF BEEF-LIVER GLUTAMATE-DEHYDROGENASE BY O-PHTHALALDEHYDE

Beef liver glutamate dehydrogenase (GDH) is inactivated by the bifunct ional reagent, o-phthalaldehyde. The initial rate of inactivation foll ows pseudo first-order kinetics. The reaction of the enzyme with o-pht halaldehyde results in isoindole derivative formation which is charact erized by typical fluorescence emission and excitation maximum at 410 nm and 337 nm, respectively. The inactivation of GDH by o-phthalaldehy de is partially prevented by alpha-ketoglutaric acid, whereas NADH doe s not provide any protection. This clearly indicates that cysteine and lysine residues are located near the alpha-ketoglutaric acid binding center. The dissociation constant of 2.2 mM was obtained for enzyme-al pha-ketoglutaric acid complex. Stoichiometry of o-phthalaldehyde bindi ng with glutamate dehydrogenase showed that the formation of approxima tely one isoindole derivative per subunit of glutamate dehydrogenase i s accompanied by complete loss of activity.