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

Document Type: Research Note

Authors

Department of Clinical Biochemistry, School of Pharmacy, Medical Sciences University of Esfahan, Esfahan, I. R. of Iran

Abstract

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.

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