12] Immobilization of nucleoside diphosphatase at the allosteric site using pyridoxal 5′-phosphate derivatives

Abstract

Nucleoside diphosphatase is an allosteric enzyme that is activated by adenosine triphosphate (ATP), other nucleoside triphosphates, and pyridoxal phosphate. Reduction of the enzyme-pyridoxal phosphate complex with NaBH 4 yields an active pyridoxyl phosphate derivative of the enzyme that is no longer sensitive to pyridoxal phosphate or ATP, but its specific activity is not significantly altered. Pyridoxal phosphate is selectively bound to the lysine residue of the allosteric site, a site different from the catalytic site of the enzyme. Sepharose-bound pyridoxal phosphate analogs have also been prepared by Ikeda and Fukui and utilized for the immobilization of vitamin B 6 -dependent enzymes, because the immobilized pyridoxal phosphate retained capacity for binding to the apoproteins. Nucleoside diphosphatase was immobilized using the immobilized pyridoxal phosphate analogs of Sepharose. The immobilized pyridoxal phosphate analog was selectively bound to the allosteric site of nucleoside diphosphatase in a way similar to free pyridoxal phosphate and active immobilized enzyme was obtained after the reduction of pyridoxal phosphate––Sepharose––enzyme complex.