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 Asp258 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. All these mutants were inactive, indicating that Asp258 may act as a p...
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Published in | The Journal of biological chemistry Vol. 269; no. 36; pp. 22642 - 22646 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
09.09.1994
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Subjects | |
Online Access | Get full text |
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Summary: | At the active site of rat prostatic acid phosphatase (rPAP), residue Asp258 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. All these mutants
were inactive, indicating that Asp258 may act as a proton donor in catalysis. Tyr123 and Arg127 residues, located at the entrance
of the active site surface in rPAP, are likely to be responsible for the substrate specificity of the enzyme. The corresponding
residues in lysosomal acid phosphatase (LAP) are Lys and Gly. In order to clarify the roles of the Tyr123 and Arg127 residues,
lysosomal type rPAP mutants (Y123K, R127G and Y123K,R127G) were generated. Sensitivity of Y123K,R127G to tartrate inhibition
was 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, unlike human LAP, the lysosomal type mutants hydrolyzed
the suggested PAP-specific substrates, phosphocreatine and phosphocholine, showing that Tyr123 and Arg127 are not the only
residues contributing to the substrate specificity of rPAP. The residues Trp106 and His112 appeared to be important in the
dimerization of rPAP. Oligomerization mutants (W106E, H112D and W106E,H112D) existed in a monomeric form without catalytic
activity or a tartrate binding ability. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(17)31694-0 |