We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solutionphase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately two orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active towards multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY−1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the Nterminal side and one or more acidic residues on the C-terminal side of pY, but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY−1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme's in vivo substrate specificity. † This work was supported by grants from the National Institutes of Health (CA132855, GM062820, CA49132, and CA49152). L.R.was supported by a predoctoral fellowship from China Scholarship Council affiliated with the Ministry of Education of P. R. China. T.M.M. was supported by a predoctoral fellowship from the NIH Chemistry-Biology Interface Training Program (T32GM08512). * To whom correspondence should be addressed: Department of Chemistry, The Ohio State University, 100 West 18 th Avenue, Columbus, OH 43210. Telephone: (614) Protein-tyrosine phosphatases (PTPs) are a large family of enzymes (the human genome encodes 107 PTPs) that catalyze the hydrolysis of phosphotyrosine (pY) in proteins to tyrosine and inorganic phosphate (1). Once thought to be promiscuous "housekeeping" enzymes, PTPs are now known to be actively involved in cell signaling and have been described as having "exquisite substrate specificity" in vivo. However, in contrast to their well-established catalytic mechanism, their physiological substrates and functions remain poorly defined. Substantial evidence suggests that the substrate specificity of PTPs is controlled by both the intrinsic sequence specificity of the catalytic domain and the presence of targeting domains, which dire...

LuxS (S-ribosylhomocysteinase) catalyzes the cleavage of the thioether linkage of S- ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor to a small signaling molecule that mediates interspecies bacterial communication called autoinducer 2 (AI-2). Inhibitors of LuxS should interfere with bacterial interspecies communication and potentially provide a novel class of antibacterial agents. In this work, SRH analogues containing substitution of a nitrogen atom for the endocyclic oxygen as well as various deoxyriboses were synthesized and evaluated for LuxS inhibition. Two of the [4-aza]SRH analogues showed modest competitive inhibition (KI ~40 μM), while most of the others were inactive. One compound that contains a hemiaminal moiety exhibited time-dependent inhibition, consistent with enzyme-catalyzed ring opening and conversion into a more potent species (KI* = 3.5 μM). The structure-activity relationship of the designed inhibitors highlights the importance of both the homocysteine and ribose moieties for high-affinity binding to LuxS active site.