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Published April 7, 1993 | Supplemental Material
Journal Article Open

Purple acid phosphatase: a diiron enzyme that catalyzes a direct phospho group transfer to water

Abstract

Purple acid phosphatases (PAPS) catalyze the hydrolysis of aryl phosphoric monoesters, phosphoric anhydrides, and phosphoproteins containing phosphoserine residues,' and these enzymes contain a mixed-valence binuclear iron center. Three mechanisms have been proposed: metal-catalyzed release of metaphosphate, direct attack of a metal-coordinated hydroxide at phosphorus, and attack by an enzyme nucleophile to produce a phosphoenzyme intermediate that is subsequently hydrolyzed. This third possibility was supported by several lines of evidence, including the observation of a "burst" of p-nitrophenol, the appearance of transphosphorylation products upon incubation of PAP with p-nitrophenyi phosphate, and retention of ^(*32)P by PAP after incubation with [γ-^(32)P]-labeled ATP. Such behavior is similar to that of other nonspecific phosphatases that form covalent phosphoenzyme intermediates. To allow us to distinguish among the three mechanistic possibilities, the stereochemistry of phospho group transfer to water was determined for the reaction catalyzed by this enzyme. We find that PAP transfers the phospho group with overall inversion of the configuration at phosphorus. This result rules out a phosphoenzyme pathway and a long-lived metaphosphate intermediate and supports the direct transfer of the phospho group to water.

Additional Information

© 1993 American Chemical Society. Received November 2, 1992. We thank the NIH for financial support (Grant GM32117 to B.A.A., GM21659 to J.R.K.). E.G.M. was a NSF predoctoral fellow. NMR spectra were recorded on instruments funded by the NIH (Grant 1-SIO-RR04870) and the NSF (Grant CHE88-14019); mass spectra were obtained at the Harvard University Chemistry Department Mass Spectrometry Facility (NIH grant 1-SIO-RR06716; NSF grant CHE-9020043).

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Supplemental Material - ja00060a055_si_001.pdf

Supplemental Material - ja2974.pdf

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