Polyphosphate kinase from Propionibacterium shermanii. Demonstration that polyphosphates are primers and determination of the size of the synthesized polyphosphate.

Abstract

Polyphosphate kinase from Propionibacterium shermanii was purified to 70% homogeneity and shown to be a monomeric enzyme of molecular weight 83,000 +/- 3,000. It was demonstrated that short chains of polyphosphate serve as primers by using [32P]polyphosphate, 6-80 residues in length for synthesis of long-chain polyphosphate glucokinase, the radiolabel was found to be at the end of the polymer, proving that the mechanism of elongation of polyphosphate by polyphosphate kinase is strictly processive. Only 1 out of 3-8 of the polyphosphate chains contained the primer, indicating that there is a second unknown pathway of initiation which does not involve the polyphosphate primer. The termination of polyphosphate synthesis was investigated. With polyphosphate as a primer, the majority of the synthesized polyphosphate was 750 residues in length. With phosphate, in place of the polyphosphate primer, the major portion was about 2,000 residues in length but there was a large span of chain lengths down to 300. Termination is influenced by pH, temperature, and the concentration of the polyphosphate primer, with the chain length decreasing as either the temperature or the concentration of primer is increased.