P-31 NMR OF ALKALINE-PHOSPHATASE - SATURATION TRANSFER AND METAL-PHOSPHORUS COUPLING

The rate constants which characterize the formation and breakdown of the noncovalent (E .cntdot. P) and covalent (E-P) enzyme-phosphate intermediates on the [Escherichia coli] alkaline phosphatase reaction pathway are sensitive to the nature of the metal ion bound to the enzyme. 31P NMR saturation transfer provides a simple and sensitive method for measuring the metal ion dependence of these rates under equilibrium conditions. When the native Zn2+ was replaced by Cd2+, the 31P NMR spectrum at high pH revealed a new resonsance at 12.6 ppm which was assigned to the noncovalent enzyme .cntdot. phosphate complex. Reconstituting the enzyme with enriched 113Cd2+ caused this unusually downfield-shifted resonance to appear as a doublet due to 113Cd-31P spin coupling (2J31P-O-113Cd = 30 Hz). This result provides the 1st unequivocal evidence for direct metal-phosphate interaction in alkaline phosphatase.