SITE-DIRECTED MUTAGENESIS OF PROSTATIC ACID-PHOSPHATASE - CATALYTICALLY IMPORTANT ASPARTIC-ACID-258, SUBSTRATE-SPECIFICITY, AND OLIGOMERIZATION

At the active site of rat prostatic acid phosphatase (rPAP), residue A sp(258) is a suitable candidate to act as an acid/base catalyst during phosphoester hydrolysis. It was changed to Asn, Ser, and Ala by site- directed mutagenesis. Ah these mutants were inactive, indicating that Asp(258) may act as a proton donor in catalysis. Tyr(123) and Arg(127) residues, located at the entrance of the active site surface in rPAP, are Likely to be responsible for the substrate specificity of the enz yme. The corresponding residues in lysosomal acid phosphatase (LAP) ar e Lys and Gly. In order to clarify the roles of the Tyr(123) and Arg(1 27) residues, lysosomal type rPAP mutants (Y123K, R127G and Y123K,R127 G) were generated. Sensitivity of Y123K,R127G to tartrate inhibition w as similar to that observed in the case of LAP, indicating that these residues might be responsible for differences in substrate specificity between the enzymes of prostatic and lysosomal origin. However, unlik e human LAP, the lysosomal type mutants hydrolyzed the suggested PAP-s pecific substrates, phosphocreatine and phosphocholine, showing that T yr(123) and Arg(127) are not the only residues contributing to the sub strate specificity of rPAP. The residues Trp(106) and His(112) appeare d to be important in the dimerization of rPAP. Oligomerization mutants (W106E, H112D and W106E,H112D) existed in a monomeric form without ca talytic activity or a tartrate binding ability.