Substrate specificity and protonation state of Escherichia coli ornithine transcarbamoylase as determined by pH studies. Binding of carbamoyl phosphate.
Binding of carbamoyl phosphate to Escherichia coli ornithine transcarbamoylase and its relation to turnover have been examined as a function of pH under steady-state conditions. The pH profile of the dissociation constant of carbamoyl phosphate (Kiacp) shows that the affinity of the substrate increases as pH decreases. Two ionizing groups are involved in carbamoyl phosphate binding. Protonation of an enzymic group with pKa 9.6 results in productive binding of the substrate with a moderate affinity of Kiacp approximately 30 microM. Protonation of a second group further enhances binding by roughly another order of magnitude. This ionization occurs with a pKa that shifts from less than 6 in the free enzyme to 7.3 in the binary complex. However, tighter binding of carbamoyl phosphate due to this ionization does not contribute to catalysis. The turnover rate (kcat) of the enzyme diminishes in the acidic pH range and is governed by an ionization with a pKa of 7.2. Both the catalytic pKa of 7.2 and the productive binding pKa of 9.6 appear in the pH profile of kcat/KMcp. Together with earlier kinetic results (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761), these data suggest that the step which modulates kcat may occur prior to the binding of the second substrate L-ornithine.
Zambidis
I
I
Department of Chemistry, Metcalf Center for Science & Engineering, Boston University, Massachusetts 02215.
Kuo
L C
LC
D002219:Carbamates / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism*; D011485:Protein Binding; D011522:Protons; D013379:Substrate Specificity
D002219:Carbamates; D011522:Protons; D002221:Carbamyl Phosphate; D009954:Ornithine Carbamoyltransferase