Purification and Properties of Bacillus subtilis Nucleoside Diphosphokinase

Abstract

Abstract Bacillus subtilis nucleoside diphosphokinase was purified 1,100-fold to apparent homogeneity (a single band upon disc gel electrophoresis). Gel filtration through Sephadex G-200 indicated an approximate molecular weight of 100,000. The corrected Michaelis constants for the various nucleoside triphosphate substrates ranged from 0.10 mm for GTP to 0.42 mm for CTP. Deoxyribonucleoside triphosphates as well as ribonucleoside triphosphates served as substrates for the enzyme. Mn2+ and Mg2+ both were able to fulfill the divalent cation requirements for enzyme activity. Mn2+ was maximally activating at 1 mm while Mg2+ was maximally activating at 10 mm. The purified enzyme reacts with [γ-32P]ATP yielding a phosphorylated enzyme in which the phosphate group is bound via a 1-phosphohistidine bond. The equilibrium between the free and phosphorylated enzyme form favors the free form, indicating that the 1-phosphohistidine bond has a free energy of hydrolysis more negative than the terminal ATP bond. Initial velocity and product inhibition kinetic studies suggest a ping pong reaction mechanism (the first product dissociates before the second substrate combines with the enzyme). This is consistent with the postulation that the phosphorylated protein associated with enzyme activity is an intermediate in the enzymatic reaction.