Phosphohistidine as a stoichiometric intermediate in reactions catalyzed by isoenzymes of wheat germ acid phosphatase. |
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Authors: | R L Van Etten M E Hickey |
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Affiliation: | Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 USA |
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Abstract: | Burst titration experiments conducted on a highly purified isoenzyme of wheat germ acid phosphatase under conditions where [S]o > Km indicate that there is one titratable active site per molecule of enzyme of molecular weight 59,000. The enzyme is labeled to only a small extent with inorganic [32P]phosphate ion. Incubation of wheat germ acid phosphatase with 32P-labeled substrates such as p-nitrophenyl phosphate or inorganic pyrophosphate followed by quenching in alkali results in the stoichiometric trapping of a base-stable, acid-labile phosphorylated protein. The extent of 32P incorporation parallels the degree of purity of the enzyme and corresponds to the incorporation of 1 mol of phosphate per mole of enzyme. The incorporation is eliminated by the simultaneous presence of excess unlabeled phosphate ion (a competitive inhibitor) and is not observed when a noncatalytic protein (such as bovine serum albumin) is substituted for the enzyme. Complete alkaline hydrolysis of the labeled protein results in the recovery of an 85% yield of τ-phosphohistidine, identified by ion-exchange chromatography, high-voltage paper electrophoresis, and comparison with a synthetic sample. A 32P-labeled tryptic tetradecapeptide was isolated following hydrolysis of the labeled, reduced, and carboxymethylated protein with trypsin at pH 8.3, separation of the labeled peptide, and purification by two methods including a novel variant of a diagonal electrophoresis technique. The end groups and composition of the peptide are reported. The data are consistent with the interpretation that a phosphohistidine-enzyme intermediate is formed as an obligatory intermediate in the catalytic reaction involving this enzyme. |
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