Miles B W, Banzon J A, Raushel F M
Department of Chemistry, Texas A&M University, College Station 77843, USA.
Biochemistry. 1998 Nov 24;37(47):16773-9. doi: 10.1021/bi982018g.
Carbamoyl phosphate synthetase catalyzes the hydrolysis of glutamine by the nucleophilic attack of an active site cysteine residue through a mechanism that requires the formation of a gamma-glutamyl thioester intermediate. The steady-state mole fraction of the thioester intermediate was determined to be 0.23 in the presence and absence of ATP and bicarbonate. The kinetics of formation and hydrolysis of the gamma-glutamyl thioester intermediate during CPS catalyzed hydrolysis of glutamine were determined. When ATP and bicarbonate are added to CPS and glutamine, the kcat for glutamine hydrolysis increases from 0.17 to 150 min-1. The observed rate constant for thioester intermediate formation increases from 18 to 580 min-1, and the microscopic rate constant for hydrolysis of the intermediate increases from 0.15 to 460 min-1. These results demonstrate the kinetic competence of the thioester intermediate during glutamine hydrolysis. The rate-determining step changes from the hydrolysis of the intermediate when ATP and bicarbonate are absent to the formation of the intermediate upon the addition of ATP and bicarbonate. The 3 order of magnitude increase in the rate of glutamine hydrolysis upon the addition of ATP and bicarbonate is indicative of the allosteric communication between two of the three reaction centers of CPS. These sites are physically separated by approximately 45 A.
氨甲酰磷酸合成酶通过活性位点半胱氨酸残基的亲核攻击催化谷氨酰胺水解，其机制需要形成γ-谷氨酰硫酯中间体。在有和没有ATP及碳酸氢盐的情况下，硫酯中间体的稳态摩尔分数被测定为0.23。测定了在氨甲酰磷酸合成酶催化谷氨酰胺水解过程中γ-谷氨酰硫酯中间体的形成和水解动力学。当向氨甲酰磷酸合成酶和谷氨酰胺中添加ATP和碳酸氢盐时，谷氨酰胺水解的催化常数（kcat）从0.17增加到150 min⁻¹。硫酯中间体形成的观测速率常数从18增加到580 min⁻¹，中间体水解的微观速率常数从0.15增加到460 min⁻¹。这些结果证明了硫酯中间体在谷氨酰胺水解过程中的动力学活性。速率决定步骤从没有ATP和碳酸氢盐时中间体的水解转变为添加ATP和碳酸氢盐后中间体的形成。添加ATP和碳酸氢盐后谷氨酰胺水解速率增加3个数量级，这表明氨甲酰磷酸合成酶三个反应中心中的两个之间存在变构通讯。这些位点在空间上相距约45埃。
