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INTERACTION OF NAD+AND NADP+WITH NATIVE AND PYRIDOXAL PHOSPHATE-MODIFIED GLUCOSE 6-PHOSPHATE DEHYDROGENASE PURIFIED FROM STREPTOMYCES AUREOFACIENS | ||
| Iranian Journal of Science | ||
| مقاله 17، دوره 28، شماره 1، شهریور 2004، صفحه 149-160 اصل مقاله (290.12 K) | ||
| نوع مقاله: Regular Paper | ||
| شناسه دیجیتال (DOI): 10.22099/ijsts.2004.2847 | ||
| نویسندگان | ||
| B. HAGHIGHI* ؛ M. AKMALY | ||
| Department of Clinical Biochemistry, Esfahan University of Medical Sciences, Esfahan, I. R of Iran | ||
| چکیده | ||
| 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. | ||
| کلیدواژهها | ||
| Glucose 6 phosphate dehydrogenase؛ streptomyces aureofaciens؛ pyridoxal phosphate | ||
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