INTERACTION OF NAD+AND NADP+WITH NATIVE AND PYRIDOXAL PHOSPHATE-MODIFIED GLUCOSE 6-PHOSPHATE DEHYDROGENASE PURIFIED FROM STREPTOMYCES AUREOFACIENS

Document Type : Regular Paper

Authors

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

Abstract

Interaction of glucose 6-phosphate dehydrogenase from S. aureofaciens with NAD+, NADP+
and glucose 6-phosphate were investigated using different fluorescent probes. Binding of NAD+, NADP+
and S-NADPH to the native enzyme quenched intrinsic protein fluorescence by 100%, 10% and 21%,
respectively, from which Kd values of NAD+ (6.5 mM), NADP+ (92.0 μM) and S-NADPH (122.0 μM)
were calculated. Binding of NAD+, NADP+ and S-NADPH to the pyridoxylated enzyme in which
pyridoxal 5`-phosphate occupied a glucose 6-phosphate site, quenched the fluorescence of the pyridoxal
group on the enzyme by 20%, 57% and 96%, respectively. Kd values for the pyridoxylated enzyme were
also calculated for NAD+ (1.0mM), NADP+ (301.0μM) and S-NADPH (151.0μM). When NAD+ was
bound to the native enzyme-S-NADPH complex, to which S-NADPH was bound to only one subunit
leaving the other free, the S-NADPH fluorescence was quenched with a 10 nm blue shift in its emission
spectrum. NADP+ binding, however, enhanced S-NADPH fluorescence. The fluorescence of S-NADPH
bound to the pyridoxylated enzyme was enhanced upon NAD+ binding with a 5 nm blue shift, while
NADP+ binding had no effect. A substrate analog, glucose 1-phosphate, inhibited the enzyme
competitively with respect to glucose 6-phosphate and uncompetitively with respect to NAD+. Binding
of NAD+ to enzyme-glucose 1-phosphate complex quenched protein fluorescence (44%) with decreasing
Kd value from 6.5 mM in the absence of glucose 1-phosphate to 2.2 mM in its presence. NADP+,
however, showed opposite effects. The data demonstrated that S.aureofaciens glucose 6-phosphate
dehydrogenase undergoes different conformational changes upon NAD+ and NADP+ binding, and
modification of glucose 6-phosphate binding site by pyridoxal 5`-phosphate pulls the enzyme in a
conformation suitable for NAD+ binding.

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