PHOSPHOENOLPYRUVATE CARBOXYLASE - ALTERATION OF CATALYTIC AND REGULATORY PROPERTIES BY SITE-DIRECTED MUTAGENESIS AND ISOLATION OF THE GENE FROM AN EXTREME THERMOPHILE

The roles of several conserved amino acid residues in phosphoenolpyruvate carboxylase (PEPC, EC4.1.1.31) of E.coli were studied by site-directed mutagenesis. Mutant enzymes H138N (His138 replaced by Asn) and R587S lost the original catalytic activity but revealed the weak activity of HCO3- -dependent hydrolysis of PEP to yield pyruvate. By the use of H138N the formation of carboxyphosphate, a postulated reaction intermediate, was demonstrated for the first time. K620S and R438C were almost insensitive to an allosteric feedback inhibitor, aspartate, and the latter showed a tendency to dissociate to dimer. Furthermore, the gene for extremely thermostable PEPC was cloned and expressed in E. coli.