Slow-Binding Inhibition of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e by Peptide Thiols: Synthesis and Spectroscopic Characterization

Peptide-derived thiols of the general structure N-mercaptoacyl-leucyl-p-nitroanilide (1a−c) were synthesized and found to be potent, slow-binding inhibitors of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potencies (KI*) of these inhibitors against AAP range from 2.5 to 57 nM exceeding that of the natural product bestatin and approaching that of amastatin. The corresponding alcohols (2a−b) are simple competitive inhibitors of much lower potencies (KI = 23 and 360 μM). These data suggest that the free thiols are involved in the formation of the E·I and E·I* complexes, presumably serving as a metal ligand. To investigate the nature of the interaction of the thiol-based inhibitors with the dinuclear active site of AAP, we have recorded electronic absorption and EPR spectra of Co(II)Co(II)-, Co(II)Zn(II)-, and Zn(II)Co(II)-AAP in the presence of the strongest binding inhibitor, 1c. Both [CoZn(AAP)] and [ZnCo(AAP)], in the presence of 1c, exhibited an absorption band centered at 320 nm characteristic of an S → Co(II) ligand−metal charge-transfer band. In addition, absorption spectra recorded between 400 and 700 nm showed changes characteristic of 1c interacting with each active-site metal ion. EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that in a given enzyme molecule, 1c interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified μ-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon 1c binding. EPR spectra of [CoCo(AAP)]-1c, [ZnCo(AAP)]-1c, and [CoZn(AAP)]-1c were also recorded at lower temperature (3.5−4.0 K) and high microwave power (50−553 mW). The observed signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at geff ≈ 6.8 that is characteristic of thiolate-Co(II) interactions. These data suggest that the thiolate moiety can bind to either of the metal ions in the dinuclear active site of AAP but does not bridge the dinuclear cluster. Compounds 1a−c are readily accessible by synthesis and thus provide a novel class of potent aminopeptidase inhibitors

Diverse Levels of Sequence Selectivity and Catalytic Efficiency of Protein-Tyrosine Phosphatases

The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences, but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >105-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >105-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3–18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A co-crystal structure of PTP1B bound with a nephrin pY1193 peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic activities with respect to PTP in vivo substrate specificity and biological functions are discussed

Identification of a Key Amino Acid of LuxS Involved in AI-2 Production in Campylobacter jejuni

Autoinducer-2 (AI-2) mediated quorum sensing has been associated with the expression of virulence factors in a number of pathogenic organisms and has been demonstrated to play a role in motility and cytolethal distending toxin (cdt) production in Campylobacter jejuni. We have initiated the work to determine the molecular basis of AI-2 synthesis and the biological functions of quorum sensing in C. jejuni. In this work, two naturally occurring variants of C. jejuni 81116 were identified, one producing high-levels of AI-2 while the other is defective in AI-2 synthesis. Sequence analysis revealed a G92D mutation in the luxS gene of the defective variant. Complementation of the AI-2− variant with a plasmid encoded copy of the wild-type luxS gene or reversion of the G92D mutation by site-directed mutagenesis fully restored AI-2 production by the variant. These results indicate that the G92D mutation alone is responsible for the loss of AI-2 activity in C. jejuni. Kinetic analyses showed that the G92D LuxS has a ∼100-fold reduced catalytic activity relative to the wild-type enzyme. Findings from this study identify a previously undescribed amino acid that is essential for AI-2 production by LuxS and provide a unique isogenic pair of naturally occurring variants for us to dissect the functions of AI-2 mediated quorum sensing in Campylobacter