The role of an active site histidine in the catalytic mechanism of aspartate transcarbamoylase.
Although the allosteric enzyme aspartate transcarbamoylase from Escherichia coli has been the focus of numerous physical and enzymological studies for over 2 decades, the catalytic mechanism is still poorly understood. There has been much speculation regarding the role of a conserved histidine residue at position 134 which recent crystallographic studies have implicated in the catalytic mechanism as a general acid or as a general base. We have used a combination of site-directed mutagenesis, 13C-isotope incorporation, and high field NMR to probe the role of His134 in the catalytic mechanism of aspartate transcarbamoylase. By comparing the wild-type catalytic trimer with that from a partially active mutant in which His134 is replaced by alanine, we have assigned the 13C resonance for His134 in the wild-type enzyme. This residue is shown to have a pK less than 6, indicating that the imidazole ring is unprotonated at pH values optimal for enzymatic activity (pH 8.0). This result eliminates the possibility of His134 participating as a general acid in the carbamoyl transfer mechanism. Since the crystallographic studies indicate that His134 is close enough to hydrogen-bond to the carbonyl of the liganded bisubstrate analog N-(phosphonacetyl)-L-aspartate, the imidazole ring would be oriented as to make it unlikely that the N1 lone pair of electrons could participate in general base catalysis. Moreover, if His134 is implicated in base catalysis, we would have expected a much greater loss of activity upon its replacement by alanine. Perhaps the role of His134 is merely to help position the carbonyl group of carbamoyl phosphate for nucleophilic attack by the alpha-amino group of aspartate.
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D013386:Succinates / Q000494:pharmacology; D019802:Succinic Acid
D013386:Succinates; D001224:Aspartic Acid; D006639:Histidine; D002221:Carbamyl Phosphate; C013195:sparfosic acid; D019802:Succinic Acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid