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The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 Å ). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an ;1000-fold reduction in affinity relative to DPP-IV WT , while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at ;14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.Keywords: DPP-IV; X-ray crystal structure; mutant; ITC; proton NMR; short, strong hydrogen bond; serine protease; saxagliptin Dipeptidyl peptidase IV is a serine protease that modulates the biological activity of specific circulating peptide hormones, chemokines, cytokines, and neuropeptides Abbreviations: ANS, 1-anilino-8-naphthalene sulfonate; DPP-IV, dipeptidyl peptidase IV; GLP-1, glucagon-like peptide-1; ITC, isothermal titration calorimetry; pNA, p-nitroaniline; P n -P n9 , amino acid residues of the substrate which numerically indicate the position relatively to the scissile bond, n being the residues toward the N terminus and n9 being the residues toward the C terminus (this nomenclature was first defined by Schechter and Berger [1967]); SEC-MALS, size-exclusion chromatography-multiple angle light scattering; SKIE, solvent kinetic isotope effect; SSHB, short strong hydrogen bond; SIHB, short ionic hydrogen bond; TSE, thermal stability enhancement.Article and publication are at http://www.proteinscience.org/cgi

The Escherichia coli MurB gene encoding UDP-N-acetylenolpyruvylglucosamine reductase was expressed to a level of approximately 100 mg/L as a fusion construct with maltose binding protein. Rapid affinity purification, proteolysis, and anion exchange chromatography yielded homogeneous enzyme containing 1 mol/mol bound FAD. Enzyme was maximally activated by K+, NH4+, and Rb+ at cation concentrations between 10 and 50 mM. Steady-state enzyme kinetics at pH 8.0 and 37 degrees C revealed weak and strong substrate inhibition by NADPH and UDP-N-acetylenolpyruvylglucosamine, respectively, where the KiS were 910 microM and 73 microM. Substrate inhibition was pH dependent for both substrates. Initial velocity measurements as a function of both substrates produced patterns consistent with a ping pong bi bi double competitive substrate inhibition mechanism. Data at pH 8.0 yielded kinetic constants corresponding to Km,UNAGEP = 24 +/- 3 microM, Ki,UNAGEP = 73 +/- 19 microM, Km,NADPH = 17 +/- 3 microM, Ki,NADPH = 910 +/- 670 microM, and kcat = 62 +/- 3 s-1. A slow anaerobic exchange reaction with thio-NADP+ provided evidence for release of NADP+ in the absence of UNAGEP. Alternate reduced nicotinamide dinucleotides, including NHXDPH, 3'-NADPH, and alpha-NADPH, were substrates, whereas NADH was not. Several nucleotides, including ADP and UDP, were weak inhibitors of the enzyme with inhibition constants between 5 and 97 mM. Various analogs of NADP+, including 3'-NADP+, thio-NADP+, APADP+, NEthDP+, and NHXDP+, were inhibitors of the enzyme with respect to NADPH and yielded inhibition constants in the range of 110-1100 microM. Analogs without the 2'- or 3'-phosphate of NADPH or NADP+ were not substrates or inhibitors. Double inhibition experiments with varied APADP+ and UNAG produced inhibition patterns consistent with mutually exclusive inhibitor binding. The data suggest that NADPH and UNAGEP share a subsite that prevents both molecules from binding at once.

Pregnane X receptor (PXR) transactivation and binding assays have been developed into high-throughput assays, which are robust and reproducible (Z′ > 0.5). For most compounds, there was a good correlation between the results of the transactivation and binding assays. EC 50 values of compounds in the transactivation assay correlated reasonably well with their IC 50 values in the binding assay. However, there were discrepancies with some compounds showing high binding affinity in the binding assay translated into low transactivation. The most likely cause for these discrepancies was an agonistdependent relationship between binding affinity and transactivation response. In general, compounds that bound to human PXR and transactivated PXR tended to be large hydrophobic molecules. (Journal of Biomolecular Screening 2004:533-540)

Initial velocity methods were used to probe the kinetic mechanism of Escherichia coli uridine diphosphate-N-acetylmuramate:L-alanine ligase (UNAM:L-Ala ligase). When the activity (in the forward direction) versus substrate concentration data were plotted in double-reciprocal form, all line patterns were intersecting. The best fit of these data was to the equation for an ordered mechanism with the following parameters: k(cat), 1000 +/- 100 min(-1); Kma, 210 +/- 40 microM; Kmb, 84 +/- 20 microM; Kmc, 70 +/- 15 microM; Kia, 180 +/- 50 microM; Kib, 68 +/- 24 microM. Initial velocity line patterns were also determined when the concentration of one substrate was varied at different fixed concentrations of a second substrate while the third substrate was held at a concentration more than 100 times its Km value. Reciprocal plots of data collected with either ATP or L-alanine present at more than 100 times their Km values resulted in intersecting line patterns. Data collected with UNAM present at 100 times its Km value gave a set of parallel lines. These data are consistent with UNAM binding as the second substrate in an ordered mechanism. ADP, uridine diphosphate-N-acetylmuramoyl-L-alanine (UNAMA), and phosphate were tested as product inhibitors versus substrates. None of the products were competitive inhibitors versus L-alanine or UNAM, while the only observed competitive inhibition was ADP versus ATP. These results are consistent with an ordered kinetic mechanism wherein ATP binds first, UNAM binds second, and ADP is the last product released. Rapid quench experiments were performed in the presence of all three substrates or in the presence of ATP and UNAM. The production of acid-labile phosphate as a function of time is characterized by a burst phase followed by a slower linear phase with the rate close to k(cat) in the presence of all three substrates. Only a burst phase was observed for the time course of the reaction in the presence of ATP and UNAM. In both cases, the burst rate was identical. These observations are consistent with L-alanine being the third substrate to bind in a sequential mechanism involving a putative acyl-phosphate intermediate.

Protease inhibitors (PIs) are highly effective drugs against the human immunodeficiency virus (HIV), yet long-term therapeutic use is limited by emergence of HIV type 1 (HIV-1) protease substitutions that confer cross-resistance to multiple protease inhibitor drugs. Atazanavir is a highly potent HIV protease inhibitor with a distinct resistance profile that includes effectiveness against most HIV-1 isolates resistant to one or two PIs. The signature resistance substitution for atazanavir is I50L, and it is frequently (53%) accompanied by a compensatory A71V substitution that helps restore viability and increases atazanavir resistance levels. We measured the binding affinities of wild-type (WT) and I50L/A71V HIV-1 proteases to atazanavir and other currently approved PIs (ritonavir, lopinavir, saquinavir, nelfinavir, indinavir, and amprenavir) by isothermal titration calorimetry. Remarkably, we find that all of the PIs have 2-to 10-fold increased affinities for I50L/A71V protease, except for atazanavir. The results are also manifested by thermal stability measures of affinity for WT and I50L/A71V proteases. Additional biophysical and enzyme kinetics experiments show I50L/A71V protease is a stable enzyme with catalytic activity that is slightly reduced (34%) relative to the WT. Computational modeling reveals that the unique resistance phenotype of I50L/A71V protease likely originates from bulky tert-butyl groups at P2 and P2 (specific to atazanavir) that sterically clash with methyl groups on residue L50. The results of this study provide a molecular understanding of the novel hypersusceptibility of atazanavir-resistant I50L/A71V-containing clinical isolates to other currently approved PIs.

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor® affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K d ϳ 50 pM, K d ϳ 2.5 M, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.

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