A Single Amino Acid Substitution Alters Substrate Sequence Specificity of the Yeast Protein Tyrosine Phosphatase YPTP1

Protein tyrosine phosphatases (PTPases) constitute a family of enzymes that catalyze the hydrolysis of a phosphate moiety from a phosphotyrosyl residue of a protein. Because of the biological importance of the reversible tyrosine phosphorylation, much research has been focused on the PTPases since its first identification in 1988. More than 70 PTPases have been identified in various organisms including human to yeasts. Much progress has been achieved in the study of the mechanism of catalysis. However, in vivo substrates and biological roles of most of the PTPases still remains to be elucidated. PTPases recognize short phosphotyrosyl(pY)-peptides as substrates and the sequence specificities of several PTPases toward pY-peptides have been investigated. We previously reported that the lowest KM substrate for the yeast PTPase, YPTP1, was DADEpYDA which is characterized by the multiple acidic residues at the N-terminal side of pY. Similar trends were also found in many other PTPases including PTP1B, HPTPβ, T-cell PTPase, PTP-5 from bovine brain and PTP-1 from rat brain, Yersinia PTPase and rat PTP1. In the case of PTP1B, the sequence specificity was explained on the molecular basis by Jia et al. X-ray crystallographic analysis of inactive PTP1B complexed with a phosphotyrosyl peptide, DADEpYL-NH2, revealed that Arg-47 is responsible for defining the sequence specificity of PTP1B; Arg-47 forms salt bridges with the carboxyl side chains of Asp and Glu at -1 and -2 position of N-terminal side of pY (pY-1 and pY-2 positions) of the peptide. It is noteworthy that rat brain PTP-1, Yersinia PTPase, and T-cell PTPase, which share a preference for pY-peptides with acidic amino acid residues N-terminal to the pY, contain Arg at the equivalent position of Arg-47 of PTP1B. YPTP1, on the other hand, has Val-59, not Arg, at the equivalent position still exhibiting sequence specificity similar to the PTPases mentioned above. HPTPβ does not have Arg at the position, either. It is Gln in HPTPβ. Diverse amino acid residues appear in PTPases at the position equivalent to Arg-47 of PTP1B; they are Ser, Lys, Val, Ile, Gln, Thr and Ala. To investigate if Val-59 is an important determinant for the peptide substrate recognition by YPTP1, we substituted Val59 with Asp or His by site-directed mutageneses and performed kinetic experiments. These mutations that change the hydrophobic side chain of Val to amino acid side chains with a negative or a positive charge might alter the preferences of YPTP1 for the peptide substrate sequences. Previously we subcloned the YPTP1 gene in the E. coli expression vector pT7-7 (pT7-YPTP1) and used it for the overexpression of the wild-type YPTP1. To generate the mutants, YPTP1 (V59D) and YPTP1 (V59H), we modified pT7-YPTP1 plasmid by the ‘‘overlap extension method’’ based on polymerase chain reaction (PCR) technique. The wild-type and mutant PTPases were purified to > 90% purity by the combination of affinity chromatography and conventional chromatography techniques as described previously for wild-type YPTP1. A few milligrams of wild-type or mutant enzymes are generally obtained from 1 liter of E. coli culture. Specific activities of purified mutant enzymes toward p-nitrophenyl phosphate are 30-40 μmol · min−1mg−1 which are essentially indistinguishable with that of wild-type YPTP1. Kinetic experiments were performed with wild-type and the two mutant phosphatases (Table 1). Chemically synthesized 5 mer pY-peptides with sequences AXApYA (X is a variable amino acid) were used as substrates. Eventhough the negatively charged residues at pY-2 and pY-1 positions are both necessary for high affinity recognition by YPTP1, to avoid complication, the -1 position is fixed to Ala and only the -2 position of pY was changed sequentially. The first substrate examined in this study was ADApYA. Toward this substrate wild-type YPTP1 exhibited a KM value of 34 μM, 8.5-fold higher than that of the lowest KM substrate DADEpYDA (KM = 4 μM). Because of the importance of the negative charge at pY-2 position of the peptide, it is anticipated that the introduction of a positively charged amino acid to that position would reduce the affinity for wild-type YPTP1 and this was truely the case; KM = 159 μM