Purification and kinetics of tyrosine-sensitive 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase from Salmonella.

Tyrosine-sensitive 3-deoxy-D-arabino-heptulosonic acid ‘7phosphate synthetase was prepared by a relatively simple procedure from an operator constitutive (tyrOc) strain of Salmonella typhimurium in highly purified, nearly homogeneous form. It had a molecular weight of approximately 100,000 and was independent of Co2f for activity. In the reaction of D-erythrose 4-phosphate with enolpyruvate phosphate labeled with IsO in the C-O-P linkage, I80 was recovered almost entirely in orthophosphate released, indicating a C-O cleavage mechanism. The kinetic properties of the enzyme were studied by measuring release of 32Pi from [32P]enolpyruvate phosphate. With sufficiently low levels of substrates a sequential mechanism was indicated, rather than a ping-pong mechanism as previously reported from other laboratories. Rate equations were derived for bireactant mechanisms in which conversion of the ternary complex of enzyme and substrates to that of enzyme and products is irreversible. Expected inhibition patterns were deduced and used to analyze inhibition data. Product inhibition patterns by orthophosphate and 3-deoxy-D-arabino-heptulosonic acid ‘7-phosphate indicated an ordered reaction in which enolpyruvate phosphate was the first substrate to bind to enzyme and orthophosphate the first product to be released. A mechanism for the reaction is suggested which is in accord with these kinetic results, as well as orthophosphate release with C-O cleavage and stereospecific addition of erythrose 4-phosphate to enolpyruvate phosphate. The kinetics of inhibition of enzyme by D-erythritol 4-phosphate indicated that it was an apparent analog of 3-deoxy-n-arabino-heptulosonate 7-phosphate rather than of erythrose 4-phosphate.