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 phosphoenolpyruv ate carboxylase (PEPC, EC4.1.1.31) of E.coli were studied by site-dire cted mutagenesis. Mutant enzymes H138N (His138 replaced by Asn) and R5 87S lost the original catalytic activity but revealed the weak activit y of HCO3- -dependent hydrolysis of PEP to yield pyruvate. By the use of H138N the formation of carboxyphosphate, a postulated reaction inte rmediate, was demonstrated for the first time. K620S and R438C were al most insensitive to an allosteric feedback inhibitor, aspartate, and t he latter showed a tendency to dissociate to dimer. Furthermore, the g ene for extremely thermostable PEPC was cloned and expressed in E. col i.