Purification and Characterization of the Low Molecular Weight Protein Tyrosine Phosphatase, Stp1, from the Fission Yeast Schizosaccharomyces pombe

Abstract

Genetic screening in fission yeast has identified a gene named stp1+ that rescues cdc25-22 [Mondesert et al. (1994) J. Biol. Chem. 269, 27996-27999]. This gene encodes a 17.4 kDa protein that is 42% identical to members of the low molecular weight protein tyrosine phosphatases (low M(r)PTPases) previously known to exist only in mammalian species. A simple and efficient purification procedure was developed to obtain the homogeneous recombinant yeast low M(r)PTPase, Stp1, in large quantities suitable for kinetic and structural studies. Authentic Stp1 was produced as judged by amino terminal protein sequencing and electrospray ionization mass spectrometry analyses. Stp1 was shown to possess intrinsic phosphatase activity toward both aryl phosphates (such as phosphotyrosine) and alkyl phosphates (such as phosphoserine). Stp1 also dephosphorylated phosphotyrosyl peptide/protein substrates. The yeast enzyme was 6-fold slower than the mammalian enzymes, which made it amenable to pre-steady-state stopped-flow spectroscopic kinetic analysis at 30 degrees C and pH 6.0. Burst kinetics was observed with Stp1 using p-nitrophenyl phosphate as a substrate, suggesting that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate. Interestingly, the bovine heart low M(r)PTPase was capable of removing phosphate groups from both phosphotyrosyl and phosphoseryl/threonyl protein substrates with comparable efficiencies. The low M(r)PTPases, like the Cdc25 family of phosphatases, may represent a new group of dual specificity phosphatases which may be involved in cell cycle control.

Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes

“…The PTP Stp1 is a member of the low-molecular weight PTP family, which uses a negatively charged cysteine nucleophile [44]. With its Cys11 nucleophile intact, we measured a dissociation constant for P i binding by Stp1 of 18 mM at pH 6.0 (Table 3; Figure S14), in reasonable agreement with a previous measurement [45].…”

Probing the Origins of Catalytic Discrimination between Phosphate and Sulfate Monoester Hydrolysis: Comparative Analysis of Alkaline Phosphatase and Protein Tyrosine Phosphatases

“…Our value for k cat / K M for the hydrolysis of the phosphate monoester, p NPP, of 2.6 × 10 4 M –1 s –1 (Table 1), agrees with previous measurements with this substrate. 14,20 This value corresponds to a rate enhancement over the uncatalyzed reaction rate constant, k w , of (2.2 × 10 14 )-fold [= ( k cat / K M )/ k w ] (Table 1). Our Stp1 preparation also exhibited p NPS hydrolysis activity, with k cat / K M = 5.3 × 10 –5 M –1 s –1 (Figure S1, Supporting Information; Table 1).…”

Cloning and Characterization of Secretory Tyrosine Phosphatases of Mycobacterium tuberculosis

“…The putative PTPs were expressed in E. coli, and after affinity purification, the proteins were characterized. It has been observed previously that several PTPs like IphP in N. commune and Stp1 in Schizosaccharomyces pombe can dephosphorylate both tyrosine and serine/threonine residues of substrates (25,38). In order to determine the substrate specificity of the PTPs from M. tuberculosis, MBP phosphorylated at either tyrosine or serine/threonine residues was used as a substrate in a dephosphorylation reaction with purified MPtpA or MPtpB protein.…”