Binding of magnesium in a mutant Escherichia coli alkaline phosphatase changes the rate-determining step in the reaction mechanism.
To investigate the role of magnesium at the M3 site in Escherichia coli alkaline phosphatase, site-specific mutagenesis was used to substitute Glu-322, a ligand of the Mg2+ with either aspartic acid (E322D) or alanine (E322A). The residual Mg2+ content of the E322D enzyme is about 16-fold lower than that of the wild-type enzyme, and both mutant enzymes exhibit extremely poor catalytic activity compared to the wild-type enzyme. Mg2+ is a strong activator of the E322D enzyme. The hydrolysis activity of the E322D enzyme maximally stimulated by Mg2+ is 60% of that of the wild-type enzyme. Under conditions that measure the sum of hydrolysis and transphosphorylation activities, the kcat of the E322D enzyme in the presence of 500 mM Mg2+ is 2.6-fold higher than the kcat observed for the wild-type enzyme. Zn2+ also activates the E322D enzyme, although it is not as strong an activator as Mg2+. Competition experiments suggest that the activation of the E322D enzyme by Mg2+ and Zn2+ results from binding of either of these metals to the same site on the enzyme. High concentrations of the substrate p-nitrophenyl phosphate inhibit the activity of the E322D enzyme; however, high concentrations of Mg2+ can overcome this inhibition. Stopped-flow experiments indicate that the rate-limiting step of the nonstimulated E322D enzyme at pH 8.0 differs from that of the wild-type enzyme and involves the breaking of the covalent bond between the enzyme and phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167.
Kantrowitz
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D000409:Alanine; D000469:Alkaline Phosphatase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D005971:Glutamates; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D001224:Aspartic Acid; C008644:nitrophenylphosphate; D018698:Glutamic Acid; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc; D000409:Alanine