COVALENT MODIFICATION AND SITE-DIRECTED MUTAGENESIS OF AN ACTIVE-SITETRYPTOPHAN OF HUMAN PROSTATIC ACID-PHOSPHATASE

Because tryptophans are found as part of the phosphate binding sites i n a number of proteins, human prostatic acid phosphatase (hPAP) was ex amined 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 (N BS) resulted in an inactivation of the enzyme and produced a character istic reduction of the protein absorbance at 280 nm. Two tryptophans p er subunit were modified, and this was accompanied by an apparently co mplete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantl y reduced as compared to the rate of tryptophan modification. After la beling 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, in cluding Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibito r tartrate in the DNPS-Cl modification studies. This is also consisten t with the location of a homologous residue in the structure of the ra t enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lowe r K-m values with several aromatic substrates, and also exhibited redu ced stability towards urea denaturation.