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• W1976454923 endingPage "3225" @default.
• W1976454923 startingPage "3207" @default.
• W1976454923 abstract "Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound ∼4 Å apart in the active site. Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers. Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays. These assays generate concentrations of inorganic phosphate (Pi) in excess of its inhibition constant (K i ≈ 1 μM). This tight binding by Pi has affected the majority of published kinetic constants. Furthermore, binding limits k cat/K m for reaction of p-nitrophenyl phosphate, the most commonly employed substrate. We describe a sensitive 32P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition. We have revisited basic mechanistic features of AP with these alkyl phosphate substrates. The results suggest that the chemical step for phosphorylation of the enzyme limits k cat/K m. The pH−rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK a of ≤5.5 in the free enzyme. An inactivating pK a of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule. Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion. The dependence of k cat/K m on the pK a of the leaving group follows a Brønsted correlation with a slope of βlg = −0.85 ± 0.1, differing substantially from the previously reported value of −0.2 obtained from data with a less sensitive assay. This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of phosphate monoesters. The new 32P-based assay employed herein will facilitate continued dissection of the AP reaction by providing a means to readily follow the chemical step for phosphorylation of the enzyme." @default.
• W1976454923 created "2016-06-24" @default.
• W1976454923 creator A5062346500 @default.
• W1976454923 creator A5076625801 @default.
• W1976454923 date "2002-02-07" @default.
• W1976454923 modified "2023-10-02" @default.
• W1976454923 title "Alkaline Phosphatase Revisited: Hydrolysis of Alkyl Phosphates" @default.
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