Involvement of Essential Lysine Residues in the Catalytic Activity of Glucose 6-phosphate Dehyrogenase Purified from Streptomyces Aureofaciens

Glucose 6phosphate 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 6phosphate. 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.