Metal dependence of the phosphate (oxygen)-water exchange reaction of Escherichia coli alkaline phosphatase. Kinetics followed by 31P(18O) NMR.
Phosphate-water oxygen exchange catalyzed by Escherichia coli alkaline phosphatase was monitored using the 18O shift on the 31P NMR signal of inorganic phosphate. Different kinetic patterns were observed with native zinc enzyme and with its cobalt analogue. For native enzyme at pH values ranging from 4.4 to 10.0, the distribution of 18O species in Pi, viz. P18O4, P18O316O,P18O216O2,P18O16O3,P16O4, with time is compatible with a kinetic scheme in which E-P, the noncovalent enzyme-phosphate complex, dissociates more rapidly than it forms the covalent complex E-P. For the cobalt enzyme at pH 6.8, the distribution of 18O species in Pi with time is different and leads to the conclusion that formation of E-P is more rapid than dissociation of Pi from E-P-A computer simulation gave good quantitative agreement with the observed distribution for the time course of the cobalt enzyme reaction when the ratio of the rate of formation of E-P to dissection of E-P was assumed to be 3 +/- 0.5.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010103:Oxygen Isotopes; D010710:Phosphates; D014867:Water
D010103:Oxygen Isotopes; D010710:Phosphates; D014867:Water; D000469:Alkaline Phosphatase