N-hydroxycarbamate is the substrate for the pyruvate kinase catalyzed phosphorylation of hydroxylamine

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.