Chemical rescue by guanidine derivatives of an arginine-substituted site-directed mutant of Escherichia coli ornithine transcarbamylase.
Escherichia coli ornithine transcarbamylase (OTCase) catalyzes the production of L-citrulline and phosphate from carbamyl phosphate and L-ornithine in L-arginine biosynthesis. We show that exogenous guanidines can restore activity to (chemically rescue) a catalytically-impaired site-directed mutant OTCase, R57G, in which glycine replaces an an active site arginine. The best rescue agent is guanidine hydrochloride, which enhances the rate of the mutant 2000-fold. The turnover number for the guanidine-rescued R57G mutant is 10% that of wild-type. The addition of guanidine to the R57G mutant has little effect on KMCP values, and the rescue effect is therefore attributed principally to an increase in kcat. Other compounds were screened as potential rescue agents, but rate enhancement is highly selective for guanidines. Not all guanidines show large increases in kcat. For a comparative series that includes guanidine and alkylguanidines, substituent size is inversely related to kcat. Brønsted analysis of guanidines with varying pKa 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 31P-NMR spectra, carbamyl phosphate-induced effects associated with wild-type OTCase are observed in the R57G mutant only in the presence of guanidine. The kinetic mechanism of the mutant is random in the presence or absence of guanidine, in contrast to the sequential ordered mechanism of the wild-type enzyme. Thus, chemical rescue of R57G by guanidine hydrochloride restores many but not all wild-type properties to the mutant enzyme.
Rynkiewicz
M J
MJ
Department of Physiology, Boston University School of Medicine, Massachusetts 02118, USA.
Seaton
B A
BA
D001120:Arginine; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry* / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012044:Regression Analysis; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D011994:Recombinant Proteins; D014508:Urea; D001120:Arginine; D009954:Ornithine Carbamoyltransferase