The discovery that phosphorylation of proteins occurs on nitrogen by particular kinases raises the question of whether a separate class of phosphoramidases also exists, or if known phosphatases carry out the hydrolysis of phosphoramidates. The phosphoramidase activity of a number of phosphatases with different catalytic motifs was studied using the substrates N-phenylphosphoramidate (N-phPAM) and phosphoryl imidazole (PIm). The phosphatases assayed were: the protein tyrosine phosphatase YopH; alkaline phosphatase; the dual-specificity phosphatase VHR; prostatic acid phosphatase, PAcP; PHPT1, the only known phosphohistidine phosphatase; and, the serine/threonine phosphatases Lambda PP and PP1. The catalytic efficiencies, kcat/KM (s-1M-1), were compared for the respective phosphoramidase and phosphatase activities for each enzyme. Ratios of catalytic efficiencies (kcat/KM)/(kcat/KM) of pNPP over PIm are: YopH - 27; AP - 4.1; VHR - 0.22; PAcP - 1.6; AP - 0.51; and PHPT1 - 0.00007. Lambda PP catalyzed hydrolysis of PIm, although kinetic constants could not be obtained. PP1 exhibited no phosphoramidase activity. The results show that most phosphatase catalytic motifs display catalytic promiscuity by cleaving both phosphoesters and phosphoramidates, but with a pronounced preference for one substrate type versus the other.

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