Room temperature phosphorescence study of phosphate binding in Escherichia coli alkaline phosphatase.
The phosphorescence spectrum and decay of Trp109 in Escherichia coli alkaline phosphatase was measured for the enzyme in 10 mM Tris/HCl, pH 7.4, at 21 degrees C. Changes in the spectrum and decay from the steady-state in response to non-covalent phosphate binding suggested a phosphate-induced alteration in the local environment surrounding Trp109 which lies buried below the active site. The seemingly inflexible structure in the region of Trp109, as judged by its very long phosphorescence lifetime, appeared unaltered when the enzyme was symmetrically bound with phosphate. However, the protein with phosphate bound to only one site displayed a marked increase in flexibility that extended over both subunits. For ratios of phosphate/enzyme (mol/mol) between 1.0 and 2.0, the observation of exponential phosphorescence decays with lifetimes that are a function of dilution provided evidence for the rapid exchange between phosphate half-saturated and fully-saturated enzymes consistent with observed enzyme turnover rates. The lifetimes under these conditions result in the calculation of a Kd for the dissociation of phosphate from the doubly occupied enzyme of 1.1 +/- 0.1 microM. The non-exponential decays at P/Ed (phosphate/dimeric enzyme) ratios less than 1.0 revealed that the exchange of phosphate between phosphate-free and half-saturated enzymes was not occurring on the timescale of the phosphorescence decay times, which implied that the half-saturated molecule cannot be contributing significantly to catalysis under steady-state conditions. The observation that the phosphorescence decay at a P/Ed ratio of 1.0 is exponential with a lifetime characteristic of the half-saturated species indicates that the binding of the first phosphate is significantly greater than the second, or that the binding exhibits negative cooperativity.
Sun
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Department of Chemistry, McGill University, Montreal, Quebec, Canada.
Kantrowitz
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ER
Galley
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WC
D000469:Alkaline Phosphatase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008163:Luminescent Measurements; D008274:Magnesium / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D013057:Spectrum Analysis; D013696:Temperature; D015032:Zinc / Q000378:metabolism
D010710:Phosphates; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc