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Kinetics of Substrate Hydrolysis by Molecular Variants of Escherichia coli Alkaline Phosphatase

From the ¹ From the Department of Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110

Two isozymes of Escherichia coli alkaline phosphatase have been compared in the stopped flow spectrophotometer with respect to the kinetics of hydrolysis of two phosphate esters. Hydrolysis of 2,4-dinitrophenyl phosphate at pH 5.5 is biphasic; release of chromophoric alcohol occurs in a rapid burst followed by a slower steady state. The two isozymes show similar burst rates and amplitudes but their steady state rates differ by a factor of 2. Hydrolysis of p-nitrophenyl phosphate at pH 8.0 shows no significant burst and the homogeneous steady state rate is essentially the same for both isozymes. These observations and the ionic strength dependence of the rates of these reaction steps support the hypothesis, proposed previously in the literature, that the molecular process which determines the steady state rate at pH 8.0 is responsible for the presteady state burst at pH 5.5. The steady state process at pH 5.5 must be a subsequent step in the catalytic mechanism.

A molecular hybrid containing one subunit of wild type alkaline phosphatase and one subunit from an inactive mutationally altered alkaline phosphatase shows one-half the normal burst amplitude at pH 5.5. All transient and steady state rates observed for the hybrid equal those seen for wild type enzyme, once corrected for the halved number of active sites inferred from the burst amplitude. This finding casts doubt on catalytic mechanisms involving alternately functioning subunits that several workers have proposed for E. coli alkaline phosphatase.

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				M. F. Hoylaerts, T. Manes, and J. L. Millan Mammalian Alkaline Phosphatases Are Allosteric Enzymes J. Biol. Chem., September 5, 1997; 272(36): 22781 - 22787. [Abstract] [Full Text] [PDF]