ESSENTIAL GLUTAMIC-ACID RESIDUE IN ESCHERICHIA-COLI PYROPHOSPHATASE .1. CHEMICAL MODIFICATION AND LOCALIZATION WITHIN THE PRIMARY STRUCTURE

Biochemistry(1992)

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Abstract
Inorganic pyrophosphatase of E. coli is rapidly and irreversibly inactivated by N-ethyl-5-phenylisoxazolium 3'-sulfonate (Woodward's reagent K). The appearance of a maximum at 340 nm in the absorption spectrum of the protein indicates that the enzyme forms an enolic ester with the inhibitor. The nonhydrolyzable substrate analog CaPP(i) partially protects the pyrophosphatase from the inhibitory effect of the reagent. A peptide isolated from the tryptic hydrolysate of the inactivated enzyme contains an amino acid residue critically important for enzymatic activity. The peptide corresponds to residues 95-104 of the pyrophosphatase and contains four dicarboxylic acid residues. Another peptide was isolated from the hydrolysate of the modified pyrophosphatase after treatment with protease V8. This peptide carries the modified amino acid, comprises a fragment of peptide 95-104 (residues 98-101), and has the structure Glu-Ala-Gly-Glu. The inactivation of E. coli pyrophosphatase with Woodward's reagent K is the consequence of selective modification at Glu-98, which appears to be the most reactive of the dicarboxylic acids located within the active center of the enzyme.
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INORGANIC PYROPHOSPHATASE,WOODWARDS REAGENT-K,CHEMICAL MODIFICATION,HPLC,PEPTIDE MASS SPECTROMETRY
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