Identification of an essential cysteine residue in pyridoxal phosphatase from human erythrocytes.
Pyridoxal-specific phosphatase purified from human erythrocytes was inactivated by a variety of thiol-specific reagents in a time- and concentration-dependent manner. The presence of pyridoxal phosphate, a substrate, or inorganic phosphate, a competitive inhibitor, protected the enzyme from inactivation. Phosphatase inactivated by disulfide reagents was reactivated by the addition of excess dithiothreitol, indicating that the inactivation was due to formation of a mixed disulfide between the reagent and a free cysteinyl residue at or near the active site of the enzyme. Incorporation of either 1 mol of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 0.6 mol of iodo[3H]acetate, or 0.6 mol of N-[3H]ethylmaleimide per mol of subunit led to complete inactivation of the enzyme. High concentration of phosphate prevented the incorporation of DTNB and iodo[3H]acetate. Amino acid analysis of carboxymethylated enzyme and DTNB titration of the denatured phosphatase indicated that there may be only 1 cysteinyl residue per subunit. Modification by iodoacetate did not affect the quaternary structure of the enzyme. The phosphatase modified by iodo[3H]acetate was subjected to trypsin digestion, and the resulting peptides were separated on a reverse phase C18 column. Two radioactive peaks were obtained and contained a peptide with the N-terminal sequence of Ala-Gln-Gly-Val-Leu-Phe-Asp-Cys(Cm)-Asp-Gly-Val-Leu-X-Asn-Gly. Most of the radioactivity was released with Cys(Cm). These results indicate that the cysteinyl residue in this sequence is at or near the active site and is essential for activity. Residues 5-12 and 15 of this peptide are identical with a sequence of a yeast alkaline p-nitrophenylphosphatase, and the peptide has little homology with other mammalian phosphatases.
Gao
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Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292.
Fonda
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D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology*; D005033:Ethylmaleimide / Q000378:metabolism; D006801:Humans; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010744:Phosphoric Monoester Hydrolases / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D007461:Iodoacetates; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; C027138:pyridoxine phosphate phosphatase; D010744:Phosphoric Monoester Hydrolases; D003545:Cysteine; D005033:Ethylmaleimide; D019807:Iodoacetic Acid