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 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.
Porvari
K S
KS
Biocenter Oulu, Finland.
Herrala
A M
AM
Kurkela
R M
RM
Taavitsainen
P A
PA
Lindqvist
Y
Y
Schneider
G
G
Vihko
P T
PT
D000135:Acid Phosphatase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D016367:Baculoviridae; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011467:Prostate / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001224:Aspartic Acid; D000135:Acid Phosphatase