Covalent modification and site-directed mutagenesis of an active site tryptophan of human prostatic acid phosphatase.
Because tryptophans are found as part of the phosphate binding sites in a number of proteins, human prostatic acid phosphatase (hPAP) was examined for the presence and the role of essential tryptophan residues. The pH dependence of the intrinsic fluorescence of hPAP resembled the kinetic pH dependence. Chemical modification by N-bromosuccinimide (NBS) resulted in an inactivation of the enzyme and produced a characteristic reduction of the protein absorbance at 280 nm. Two tryptophans per subunit were modified, and this was accompanied by an apparently complete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantly reduced as compared to the rate of tryptophan modification. After labeling the protein with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), two tryptic peptides containing DNPS-labeled tryptophans were isolated and the sequences were identified by amino acid sequence analysis and mass spectroscopy. One peptide corresponded to residues 172-176, and included Trp174. The other corresponded to the C-terminal sequence, including Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibitor tartrate in the DNPS-Cl modification studies. This is also consistent with the location of a homologous residue in the structure of the rat enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lower Km values with several aromatic substrates, and also exhibited reduced stability towards urea denaturation.
Zhang
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Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393, USA.
Ostanin
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Van Etten
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D000135:Acid Phosphatase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001974:Bromosuccinimide; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011467:Prostate / Q000201:enzymology; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D017931:DNA Primers; D014364:Tryptophan; D000135:Acid Phosphatase; D001974:Bromosuccinimide