Phosphoserine alkaline phosphatase

Other compounds, not shown in Table VI, which are hydrolyzed by alkaline phosphatase are TPN (28), poly A (28), phosphocellulose (28), pyrophosphoserine (102, 103), phosphoserine (102-104), pyridoxine phosphate (104), pyridoxal phosphate (104), phosphothreonine (104), and phosphocholine (104) These compounds are all hydrolyzed at approximately the same rate. 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

This feature has been extensively investigated by Engstrom (20, 71, 88, 168, 169 see also Sections I,A and II,B) whose results may be summarized as follows (1) incubation of alkaline phosphatase with 32P-Pi at pH 4—6 and 0°, followed by acid inactivation, leads to the appearance of the label in the enzyme protein (2) after acid hydrolysis the only labeled amino acid found is phosphoserine (3) one mole of Pi is incorporated per mole of enzyme (4) the presence of Zn2+ in the enzyme is essential for phosphorylation (5) bound Pi can be displaced by addition of glucose 6-phosphate to the phosphorylation medium and (6) very little phosphoryl enzyme is formed under alkaline conditions. 

Dephosphorylation. In vivo, phosphoprotein phosphatases (phos-phoprotein phosphohydrolase EC 3.1.3.16) participate in the regulation of phosphate turnover and in rapid reversal of the phosphorylating reactions, yet there is very little information on this important group of enzymes. Alkaline (104) and acid (105) phosphatases of milk can hydrolyze the phosphate groups of phosphoserine residues in the caseins. E. coli alkaline phosphatase dephosphorylates many phosphoproteins (106,107). A phosphoprotein phosphatase of liver dephosphorylates phosphorylated histones and protamines but has little or no activity on casein or phosvitin (108). The 60-fold purified enzyme had an apparent Km for dephosphorylation of histone I (FI) of 2 X 10"5M and a pH optimum of 7-8. Histone phosphatase activity was detected in all eukaryotic cells examined, but it was not found in the extracts of several prokaryotes. 

Fig. 12. P-NMR spectra (proton-decoupled, 109.3 MHz) of hen egg-white ovalbumin at pH 8.3. Assignments of the resonances (a) phosphoserine-68, 5.0 ppm, and phosphoserine-344, 4.75 ppm (b) same as in (a) but 1 h after the addition of 15 mAf MgCl2 (causes a small upheld shift) and E. coli alkaline phosphatase (c) same as in (b) but 20 h after the addition of the phosphatase. The new resonance appearing at 3 ppm is inorganic phosphate liberated by the phosphatase treatment. From Vogel and Bridger (1982c). Copyright 1982 American Chemical Society.

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