Weiss P M, Hermes J D, Dougherty T M, Cleland W W
Biochemistry. 1984 Sep 11;23(19):4346-50. doi: 10.1021/bi00314a015.
The true substrate for the pyruvate kinase catalyzed phosphorylation of hydroxylamine at high pH which is activated by bicarbonate is shown to be N-hydroxycarbamate, since a lag is seen when the reaction is started by the addition of bicarbonate or hydroxylamine but a burst appears when it is started with a mixture of the two. The lag can be diminished by addition of carbonic anhydrase but not eliminated, showing that CO2 is an intermediate in the formation of the carbamate and that both the formation of CO2 and the subsequent reaction of CO2 with hydroxylamine limit the rate of carbamate formation. The equilibrium constant for the reaction bicarbonate + hydroxylamine reversed N-hydroxycarbamate is 1.33 M-1. The product of the phosphorylation decomposes by loss of CO2 to O-phosphorylhydroxylamine, which is stable at 25 degrees C between pH 3 and 11 and has pK2 = 5.63 for the phosphate and pK3 = 10.26 for the amino group.
在高pH值下，由丙酮酸激酶催化、被碳酸氢盐激活的羟胺磷酸化反应的真正底物被证明是N - 羟基氨基甲酸盐。因为当通过添加碳酸氢盐或羟胺启动反应时会出现延迟，但当用两者的混合物启动反应时会出现爆发。添加碳酸酐酶可减少延迟，但不能消除，这表明CO₂是氨基甲酸盐形成过程中的中间体，并且CO₂的形成以及随后CO₂与羟胺的反应都限制了氨基甲酸盐的形成速率。碳酸氢盐 + 羟胺 可逆反应 N - 羟基氨基甲酸盐的反应平衡常数为1.33 M⁻¹。磷酸化产物通过失去CO₂分解为O - 磷酰羟胺，其在25℃下于pH 3至11之间稳定，磷酸盐的pK₂ = 5.63，氨基的pK₃ = 10.26。
