CHEMICAL RESCUE BY GUANIDINE DERIVATIVES OF AN ARGININE-SUBSTITUTED SITE-DIRECTED MUTANT OF ESCHERICHIA-COLI ORNITHINE TRANSCARBAMYLASE

Escherichia coli ornithine transcarbamylase (OTCase) catalyzes the pro duction of L-citrulline and phosphate from carbamyl phosphate and L-or nithine in L-arginine biosynthesis, We show that exogenous guanidines can restore activity to (chemically rescue) a catalytically-impaired s ite-directed mutant OTCase, R57G, in which glycine replaces an an acti ve site arginine, The best rescue agent is guanidine hydrochloride, wh ich enhances the rate of the mutant 2000-fold. The turnover number fur the ru guanidine-rescued R57G mutant is 10% that of wild-type, The ad dition of guanidine to the R57G mutant has little effect on K-M(CP) va lues, and the rescue effect is therefore attributed principally to an increase in k(cat). Other compounds were screened as potential rescue agents, but rate enhancement is highly selective for guanidines. Not a ll guanidines show large increases in k(cat). For a comparative series that includes guanidine and alkylguanidines, substituent size is inve rsely related to k(cat). Bronsted analysis of guanidines with varying pK(a) values indicates that a partial positive charge is implicated in rescue, consistent with the proposed role of arginine 57 in catalysis . In UV difference and P-31-NMR spectra, carbamyl phosphate-induced ef fects associated with wild-type OTCase are observed in the R57G mutant only in the presence of guanidine. The kinetic mechanism of the mutan t is random in the presence or absence of guanidine, in contrast to th e sequential ordered mechanism of the wild-type enzyme, Thus, chemical rescue of R57G by guanidine hydrochloride restores many but not all w ild-type properties to the mutant enzyme.