Efforts to further elucidate structure-activity relationships (SAR) within our previously disclosed series of beta-quaternary amino acid linked l-cis-4,5-methanoprolinenitrile dipeptidyl peptidase IV (DPP-IV) inhibitors led to the investigation of vinyl substitution at the beta-position of alpha-cycloalkyl-substituted glycines. Despite poor systemic exposure, vinyl-substituted compounds showed extended duration of action in acute rat ex vivo plasma DPP-IV inhibition models. Oxygenated putative metabolites were prepared and were shown to exhibit the potency and extended duration of action of their precursors in efficacy models measuring glucose clearance in Zucker(fa/fa) rats. Extension of this approach to adamantylglycine-derived inhibitors led to the discovery of highly potent inhibitors, including hydroxyadamantyl compound BMS-477118 (saxagliptin), a highly efficacious, stable, and long-acting DPP-IV inhibitor, which is currently undergoing clinical trials for treatment of type 2 diabetes.

The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In order to quantitatively assess the catalytic capabilities of the enzyme, the native catalase-peroxidase from Mycobacterium smegmatis was purified over 200-fold to homogeneity. The enzyme was shown to exhibit both catalase and peroxidase activities, and in the presence of either hydrogen peroxide or t-butyl peroxide, was found to catalyze the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as well as artificial peroxidase substrates, at rates between 2.7 and 20 s ؊1 . The homogeneous enzyme exhibited a visible absorbance spectrum typical of ferric heme-containing catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature (10 K) electron paramagnetic resonance spectra in the presence of ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t1 ⁄2 ‫؍‬ ϳ 20 min) reduced by dithionite, and the reduced form showed typical spectral changes when either KCN or CO were subsequently added. The M. smegmatis catalase-peroxidase was found to contain 2 heme molecules per tetramer, which were identified as iron protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic activity was inhibited by KCN, NaN 3 , isoniazid (isonicotinic acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by 3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases in the oxidative activation of the antitubercular prodrug isoniazid is discussed.Metabolism in an oxygen-containing environment results in the generation of reactive oxygen species such as superoxide, hydroxyl radical, and hydrogen peroxide. Organisms have evolved sophisticated and efficient enzyme systems to neutralize these potentially injurious reactive oxygen species, including catalases, superoxide dismutases, and peroxidases. Enzymes that exhibit either catalatic or peroxidatic activity are common among animals, plants, and microorganisms, while bacteria and some fungi have been found to contain hydroperoxidases that exhibit both catalase and peroxidase activities. These catalase-peroxidases have been purified from Escherichia coli B (1), Klebsiella pneumoniae (2), Halobacterium halobium (3), Septoria tritici (4), and a Bacillus strain YN-2000 (5), and exhibit visible absorbance spectra typical of a ferric high spin hemoprotein. The pyridine ferrohemochromes of these enzymes show an absorption spectrum typical of ferric protoheme IX (1-5). A characteristic feature of the bacterial catalase-peroxidases is their low heme content, with only 2 molecules of heme per enzyme tetramer. The activity of bacterial catalase-peroxidases is inhibited by azide and cyanide, but not by the catalase-specific inhibitor, 3-amino-1,2,4-triazole.The enzymological characterization of mycobacterial catalase-peroxidases is relevant for a number of reasons. Human mycobacterial pathogens, such as Mycobacterium tuberculosis, Myc...

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

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.

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Antisense RNA is a versatile tool for reducing gene expression. It was used to determine if ahpC, a gene that is involved in defence against oxidative stress and isoniazid (INH) resistance, is important for virulence of Mycobacterium bovis, a member of the Mycobecterium tuberculosis complex. Antisense RNA constructs o f ahpC were made using different strength promoters in front of a reversed coding sequence of ahpC. These constructs were electroporated into a virulent wild-type M. bowis strain and a moderately virulent INH-resistant M. bo wis strain that was cata lase/peroxi dase-negat ive. Down-regulation of protein synthesis occurred and this was visualized by immunoblotting. All strains containing antisense RNA were markedly less virulent than their parent strains in guinea pigs. M. bowis with an up-regulated ahpC gene was more resistant t o cumene hydroperoxide than its parent strain, which had a wildtype ahpC promoter. These results agree with a model of INH resistance in which overexpression of AhpC compensates in some INH-resistant strains for loss of catalase/peroxidase by maintaining the ability to defend against oxidative stress mediated through organic peroxides. In addition, normal expression of AhpC is crucial for maintaining the virulence of wild-type M. bovis, which has normal catalase/peroxidase levels.

The steady-state kinetic mechanism of ␤-amyloid precursor protein-cleaving enzyme (BACE)-catalyzed proteolytic cleavage was evaluated using product and statine-(Stat(V)) or hydroxyethylene-containing (OM99-2) peptide inhibition data, solvent kinetic isotope effects, and proton NMR spectroscopy. The noncompetitive inhibition pattern observed for both cleavage products, together with the independence of Stat(V) inhibition on substrate concentration, suggests a uni-bi-iso kinetic mechanism. According to this mechanism, the enzyme undergoes multiple conformation changes during the catalytic cycle. If any of these steps are rate-limiting to turnover, an enzyme form preceding the rate-limiting conformational change should accumulate. An insignificant solvent kinetic isotope effect (SKIE) on k cat /K m , a large inverse solvent kinetic isotope effect on k cat , and the absence of any SKIE on the inhibition onset by Stat(V) during catalysis together indicate that the ratelimiting iso-step occurs after formation of a tetrahedral intermediate. A moderately short and strong hydrogen bond (at ␦ 13.0 ppm and of 0.6) has been observed by NMR spectroscopy in the enzyme-hydroxyethylene peptide (OM99-2) complex that presumably mimics the tetrahedral intermediate of catalysis. Collapse of this intermediate, involving multiple steps and interconversion of enzyme forms, has been suggested to impose a rate limitation, which is manifested in a significant SKIE on k cat . Multiple enzyme forms and their distribution during catalysis were evaluated by measuring the SKIE on the noncompetitive (mixed) inhibition constants for the C-terminal reaction product. Large, normal SKIE values were observed for these inhibition constants, suggesting that both kinetic and thermodynamic components contribute to the K ii and K is expressions, as has been suggested for other iso-mechanism featuring enzymes. We propose that a conformational change related to the reprotonation of aspartates during or after the bond-breaking event is the rate-limiting segment in the catalytic reaction of ␤-amyloid precursor protein-cleaving enzyme, and ligands binding to other than the ground-state forms of the enzyme might provide inhibitors of greater pharmacological relevance.Extracellular amyloid deposits in brain, a characteristic feature of Alzheimer's disease, is a result of proteolytic cleavage of membrane-bound amyloid precursor protein by two enzymes, ␤-secretase and ␥-secretase. The second cleavage activity (␥-secretase) is strongly associated with the presenilin multisubunit complexes (1), whereas ␤-secretase (BACE) 1 has been identified as a novel transmembrane aspartyl protease (2-4).Although aspartyl proteases have been studied for more than 4 decades, new aspects of catalysis and inhibition continue to emerge. A substantial number of these enzymes have been identified as useful targets for chemotherapeutic intervention in human diseases (5-8), yet there has been limited success in identifying clinically relevant inhibitors; hence, it is important to explo...

Isoniazid (INH) is a highly effective drug used in the treatment and prophylaxis of Mycobacterium tuberculosis infections. Resistance to INH in clinical isolates has been correlated with mutations in the inhA, katG, andahpC genes. In this report, we describe a new mechanism for INH resistance in Mycobacterium smegmatis. Mutations that reduce NADH dehydrogenase activity (Ndh; type II) cause multiple phenotypes, including (i) coresistance to INH and a related drug, ethionamide; (ii) thermosensitive lethality; and (iii) auxotrophy. These phenotypes are corrected by expression of one of two enzymes: NADH dehydrogenase and the NADH-dependent malate dehydrogenase of theM. tuberculosis complex. The genetic data presented here indicate that defects in NADH oxidation cause all of the mutant traits and that an increase in the NADH/NAD+ ratio confers INH resistance.