CATALYTIC ROLE OF AN ARGININE RESIDUE IN THE HIGHLY CONSERVED AND UNIQUE SEQUENCE OF PHOSPHOENOLPYRUVATE CARBOXYLASE

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] has a highly cons erved and unique sequence, 578-FHGRGGSIGRGGAP-591 (on Escherichia coli , PEPC), in which a GRGG motif is repeated twice with two intervening residues. Since previous chemical modification studies suggested the f unctional importance of arginine residues, the invariant Arg587 in thi s region was replaced with Ser, and the enzymatic properties of the re sulting mutant enzyme (R587S) were investigated. Replacement led to vi rtual loss of the catalytic activity to form oxaloacetate. The specifi c activity was 37 nmol . min(-1). mg(-1), which corresponds to 2 X 10( -4)-fold the activity of the wild-type enzyme. However, the activity o f bicarbonate- and Mg2+-dependent hydrolysis of phosphoenolpyruvate (P EP) to pyruvate appeared for the mutant enzyme with a specific activit y of 2.1 mu mol . min(-1). mg(-1). In view of the stepwise reaction me chanism proposed for PEPC, this activity can be attributed to impairme nt of the subsequent partial reaction(s) following the formation of th e intermediate carboxyphosphate. The half-saturation concentration (S- 0.5) of HCO3- in R587S was about 100-fold that in the wild-type enzyme , whereas the respective values for PEP and Mg2+ were 20- and 15-fold, indicative of this residue participating in the binding of HCO3-.