HETEROLOGOUS EXPRESSION OF HUMAN PROSTATIC ACID-PHOSPHATASE AND SITE-DIRECTED MUTAGENESIS OF THE ENZYME ACTIVE-SITE

Earlier covalent modification experiments indicated that histidine, arginine, and carboxylic acid residues were involved in the catalytic mechanism of the acid phosphatase enzyme from human prostate. The present study utilizes site-directed mutagenesis to evaluate the catalytic importance of 7 residues of human prostatic phosphatase, namely His(12), His(257), Asp(258), Arg(11), Arg(15), Arg(54), and Arg(79), which are highly conserved in the sequences of high molecular weight acid phosphatases. Both wild type and mutant enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions to a leader peptide of prepro-alpha-factor, which resulted in the secretion of processed proteins into the culture media. The kinetic parameters of the recombinant wild type enzyme were shown to be effectively identical with those of the native protein. The circular dichroism spectra of wild type and mutant proteins were effectively superimposable. Replacement of His(12), which was previously shown to be transiently phosphorylated during catalysis, as well as the adjacent Arg(11), both resulted in >10(4) reductions of enzyme activity. Consistent with a postu lated involvement of arginine residues in substrate binding, large reductions in V-max caused by the R15A and R79A mutations were accompanied by marked increases in K-m. However, the mutation R54A had only small effects on kinetic parameters. Essential roles for both His(257) and Asp(258) were also established. The D258A mutation not only caused larger decreases of V-max toward p-nitrophenyl phosphate compared to the effect of the H257A mutation, but it also caused the reaction to become more sensitive to the nature of the leaving group. It is concluded that His(12) and Asp(258) are involved in the formation of the phosphoenzyme intermediate as an acceptor of the phospho group and a proton donor for the substrate leaving group, respectively, while His(257) may participate in substrate binding or may facilitate the breakdown of the phosphoenzyme by maintaining the protonated state of the phosphorylated His(12) residue.