Purification and properties of phosphoribosyl‐diphosphate synthetase from Bacillus subtilis

Abstract

Phosphoribosyl‐diphosphate (PPRibP) synthetase from Bacillus subtilis has been purified to near homogeneity from an Escherichia coli Δprs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthetase, on a plasmid. The Mr of the subunit (34000) and its amino‐terminal amino acid sequence (14 residues) were in complete agreement with expectations from the nucleotide sequence of the prs gene. The Mr of the native enzyme (280000 ± 10000) was consistent with an octameric quaternary structure. No tendency toward multiple states of aggregation of the enzyme was seen. The purified enzyme required Mg2+ and inorganic phosphate for activity; Mn2+ supported only 30% the activity seen with Mg2+. Michaelis constants for ATP and ribose 5‐phosphate (Rib5P) were 0.66 mM and 0.48 mM, respectively. Of several end products tested, only ADP was strongly inhibitory; GDP was a weak inhibitor. ADP inhibition displayed homotropic cooperativity and was enhanced by increasing saturation of the enzyme with ATP. These observations strongly suggest a specific allosteric site for ADP binding. A comparison of physical and kinetic properties of bacterial and mammalian PPRibP synthetases is presented. Copyright © 1990, Wiley Blackwell. All rights reserved