INVOLVEMENT OF ESSENTIAL LYSINE RESIDUES IN THE CATALYTIC ACTIVITY OF GLUCOSE 6-PHOSPHATE DEHYROGENASE PURIFIED FROM STREPTOMYCES AUREOFACIENS

Glucose 6- phosphate dehydrogenase from streptomyces aureofaciens was purified and inactivated by pyridoxal 5?-phosphate (PLP). The inactivation was a pseudo-first order and time-dependent reaction. Complete inactivation was achieved at 0.2mM PLP within 16 minutes. The type of inhibition was competitive with respect to Glucose 6- phosphate. Spectral characteristics of PLP-enzyme complex corresponded to the formation of a Schiff’s base between PLP and lysine residue(s) of the enzyme. Intrinsic protein fluorescence sharply decreased upon PLP modification with about a 10 nm red shift. The presence of glucose 6-phosphate in the incubation mixture prevented the fluorescence change. Fluorescence studies revealed that NAD+ and NADP+ binding induces different conformational changes in pyridoxylated enzyme. The stochiometry of PLP binding to the enzyme showed that 2 moles of lysine residues were modified per mole of enzyme. The data indicated that the modified lysine residues are involved in substrate binding and/or catalytic activity of this enzyme.