interaction of nad+and nadp+with native and pyridoxal phosphate-modified glucose 6-phosphate dehydrogenase purified from streptomyces aureofaciens

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 whichpyridoxal 5`-phosphate occupied a glucose 6-phosphate site, quenched the fluorescence of the pyridoxalgroup on the enzyme by 20%, 57% and 96%, respectively. kd values for the pyridoxylated enzyme werealso calculated for nad+ (1.0mm), nadp+ (301.0μm) and s-nadph (151.0μm). when nad+ wasbound to the native enzyme-s-nadph complex, to which s-nadph was bound to only one subunitleaving the other free, the s-nadph fluorescence was quenched with a 10 nm blue shift in its emissionspectrum. nadp+ binding, however, enhanced s-nadph fluorescence. the fluorescence of s-nadphbound to the pyridoxylated enzyme was enhanced upon nad+ binding with a 5 nm blue shift, whilenadp+ binding had no effect. a substrate analog, glucose 1-phosphate, inhibited the enzymecompetitively with respect to glucose 6-phosphate and uncompetitively with respect to nad+. bindingof nad+ to enzyme-glucose 1-phosphate complex quenched protein fluorescence (44%) with decreasingkd 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-phosphatedehydrogenase undergoes different conformational changes upon nad+ and nadp+ binding, andmodification of glucose 6-phosphate binding site by pyridoxal 5`-phosphate pulls the enzyme in aconformation suitable for nad+ binding.