Meek T D, Karsten W E, DeBrosse C W
Biochemistry. 1987 May 5;26(9):2584-93. doi: 10.1021/bi00383a026.
The kinetic mechanism of carbamoyl-phosphate synthetase II from Syrian hamster kidney cells has been determined at pH 7.2 and 37 degrees C. Initial velocity, product inhibition, and dead-end inhibition studies of both the biosynthetic and bicarbonate-dependent adenosinetriphosphatase (ATPase) reactions are consistent with a partially random sequential mechanism in which the ordered addition of MgATP, HCO3-, and glutamine is followed by the ordered release of glutamate and Pi. Subsequently, the binding of a second MgATP is followed by the release of MgADP, which precedes the random release of carbamoyl phosphate and a second MgADP. Carbamoyl-phosphate synthetase II catalyzes beta gamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-18O]ATP in both the ATPase and biosynthetic reactions. Negligible exchange is observed in the strict absence of HCO3- (and glutamine or NH4+). The ratio of moles of MgATP exchanged to moles of MgATP hydrolyzed (nu ex/nu cat) is 0.62 for the ATPase reaction, and it is 0.39 and 0.16 for the biosynthetic reaction in the presence of high levels of glutamine and NH4+, respectively. The observed positional isotope exchange is suppressed but not eliminated at nearly saturating concentrations of either glutamine or NH4+, suggesting that this residual exchange results from either the facile reversal of an E-MgADP-carboxyphosphate-Gln(NH4+) complex or exchange within an E-MgADP-carbamoyl phosphate-MgADP complex, or both. In the 31P NMR spectra of the exchanged [gamma-18O]ATP, the distribution patterns of 16O in the gamma-phosphorus resonances in all samples reflect an exchange mechanism in which a rotationally unhindered molecule of [18O3, 16O]Pi does not readily participate. These results suggest that the formation of carbamate from MgATP, HCO3-, and glutamine proceeds via a stepwise, not concerted mechanism, involving at least one kinetically competent covalent intermediate, such as carboxyphosphate.
已在pH 7.2和37℃条件下测定了叙利亚仓鼠肾细胞中氨甲酰磷酸合成酶II的动力学机制。对生物合成反应和碳酸氢盐依赖性腺苷三磷酸酶（ATP酶）反应进行的初速度、产物抑制和终产物抑制研究均符合部分随机顺序机制，即按顺序依次添加MgATP、HCO₃⁻和谷氨酰胺，随后依次释放谷氨酸和磷酸根离子（Pi）。随后，第二个MgATP结合后释放MgADP，这先于氨甲酰磷酸和第二个MgADP的随机释放。氨甲酰磷酸合成酶II在ATP酶反应和生物合成反应中均催化[γ-¹⁸O]ATP的βγ桥:β非桥位置氧交换。在严格不存在HCO₃⁻（以及谷氨酰胺或NH₄⁺）的情况下，观察到的交换可忽略不计。对于ATP酶反应，交换的MgATP摩尔数与水解的MgATP摩尔数之比（νex/νcat）为0.62，在高浓度谷氨酰胺和NH₄⁺存在下，生物合成反应的该比值分别为0.39和0.16。在谷氨酰胺或NH₄⁺接近饱和浓度时，观察到的位置同位素交换受到抑制但未消除，这表明这种残余交换是由于E-MgADP-羧基磷酸-Gln(NH₄⁺)复合物的轻易逆转或E-MgADP-氨甲酰磷酸-MgADP复合物内的交换，或两者兼而有之。在交换后的[γ-¹⁸O]ATP的³¹P NMR光谱中，所有样品中γ-磷共振中¹⁶O的分布模式反映了一种交换机制，其中旋转不受阻碍的[¹⁸O₃,¹⁶O]Pi分子不易参与。这些结果表明，由MgATP、HCO₃⁻和谷氨酰胺形成氨基甲酸酯是通过逐步而非协同机制进行的，涉及至少一种动力学上可行的共价中间体，如羧基磷酸。
