Allosteric Dominance in Carbamoyl Phosphate Synthetase

• Published in: Biochemistry (Easton) 1999-02, Vol.38 (5), p.1394-1401
• Main Authors: Braxton, B. L, Mullins, Leisha S, Raushel, Frank M, Reinhart, Gregory D
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Diphosphate - metabolism; Allosteric Regulation; Allosteric Site; Binding, Competitive; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - chemistry; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - metabolism; Escherichia coli; Escherichia coli - enzymology; Inosine Monophosphate - chemistry; Inosine Monophosphate - metabolism; Ligands; Ornithine - chemistry; Ornithine - metabolism; Uridine Monophosphate - chemistry; Uridine Monophosphate - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi982097w

A linked-function analysis of the allosteric responsiveness of carbamoyl phosphate synthetase (CPS) from E. coli was performed by following the ATP synthesis reaction at low carbamoyl phosphate concentration. All three allosteric ligands, ornithine, UMP, and IMP, act by modifying the affinity of CPS for the substrate MgADP. Individually ornithine strongly promotes, and UMP strongly antagonizes, the binding of MgADP. IMP causes only a slight inhibition at 25 °C. When both ornithine and UMP were varied, models which presume a mutually exclusive binding relationship between these ligands do not fit the data as well as does one which allows both ligands (and substrate) to bind simultaneously. The same result was obtained with ornithine and IMP. By contrast, the actions of UMP and IMP together must be explained with a competitive model, consistent with previous reports that UMP and IMP bind to the same site. When ornithine is bound to the enzyme, its activation dominates the effects when either UMP or IMP is also bound. The relationship of this observation to the structure of CPS is discussed.