title: Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.
abstract: The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720.
authors.forename: J L
authors.initials: JL
authors.lastname: Turnbull
authors.forename: G L
authors.initials: GL
authors.lastname: Waldrop
authors.forename: H K
authors.initials: HK
authors.lastname: Schachman
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000378:metabolism; D002250:Carbon Radioisotopes; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002250:Carbon Radioisotopes; D007477:Ions; D011994:Recombinant Proteins; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid