Reactive groups in mammalian alkaline phosphatase: loss of enzyme activity after reaction with phenylglyoxal and iodoacetamide.

Abstract

Recently we obtained evidence for an arginine residue at the active centre of pig kidney alkaline phosphatase (Woodroofe & Butterworth, 1979). Such a residue has been implicated in Escherichia coli alkaline phosphatase and in many other enzymes that act on substrates containing a phosphate group (Daemen & Riordan, 1974; Borders & Riordan, 1975). In continuing our study of essential residues in mammalian alkaline phosphatase, we have been working with the calf intestinal enzyme, since it is available commercially in a highly purified form and its amino-acid composition has been reported (Fosset et al., 1974). Calf intestinal. alkaline phosphatase (obtained from the Boehringer Corporation) was dialysed against 0.05 M-Tris/HCI buffer, p H 8.6, before chromatography on DEAE-cellulose by the method described by Fosset et al. (1974). The purified enzyme was stored at -2OOC. For reaction with O-SOmMphenylglyoxal, 1-2pg of alkaline phosphatase was preincubated at 20°C in 0.125 M-NaHCO,, . pH 8.35. The total reaction volume was 1 cm3. At appropriate times, samples (50pl) were withdrawn and assayed for residual phosphatase activity at pH 10 (0.1 M-Na,CO,/NaHCO, buffer) and 3OoC with 2.5 mM-p-nitrophenyl phosphate as substrate. The protective action of P, at concentrations up to 2 0 0 m ~ was tested by the inclusion in the preincubation mixture of phosphate that had been adjusted to pH8.35. For inactivation by iodoacetamide, a very similar procedure was adopted, except that the preincubation was usually carried out at p H 9 (0.1 M-Na,CO,/NaHCO, buffer). The effect of the H+ concentration on the rate of inactivation was investigated by varying the pH of the preincubation mixture between 8 and 10.5. Fig. 1 shows that the intestinal enzyme is inactivated by both phenylglyoxal and iodoacetamide. P, protected the enzyme against phenylglyoxal, but has no effect on the rate of inactivation by iodoacetamide. Comparison of the rates of inactivation of alkaline phosphatase suggests that iodoacetamide is more effective than phenylglyoxal on a concentration basis. The rate of inactivation by iodoacetamide is pH-independent between 8 and 9.5, but then decreases markedly above this range. Iodoacetate also inactivates the enzyme, but requires a concentration that is approximately five times higher than those shown for iodoacetamide. N-ethylmaleimide did not inactivate the enzyme. The inactivation by phenylglyoxal and iodoacetamide could not be reversed by passage through a column of Sephadex G-25, neither was reversal achieved by treatment with /3-mercaptoE e x