A pre-steady state and steady state kinetic analysis of the N-ribosyl hydrolase activity of hCD157.

hCD157 catalyzes the hydrolysis of nicotinamide riboside (NR) and nicotinic acid riboside (NAR). The release of nicotinamide or nicotinic acid from NR or NAR was confirmed by spectrophotometric, HPLC and NMR analyses. hCD157 is inactivated by a mechanism-based inhibitor, 2'-deoxy-2'-fluoro-nicotinamide arabinoside (fNR). Modification of the enzyme during the catalytic cycle by NR, NAR, or fNR increased the intrinsic protein fluorescence by approximately 50%. Pre-steady state and steady state data were used to derive a minimal kinetic scheme for the hydrolysis of NR. After initial complex formation a reversible step (360 and 30s(-1)) is followed by a slow irreversible step (0.1s(-1)) that defined the rate limiting step, or kcat. The calculated KMapp value for NR in the hydrolytic reaction is 6nM. The values of the kinetic constants suggest that one biological function of cell-surface hCD157 is to bind and slowly hydrolyze NR, possibly converting it to a ligand-activated receptor. Differences in substrate preference between hCD157 and hCD38 were rationalized through a comparison of the crystal structures of the two proteins. This comparison identified several residues in hCD157 (F108 and F173) that can potentially hinder the binding of dinucleotide substrates (NAD+).

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates.

ADP-ribosyl cyclase and NAD+ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD+, nicotinamide adenine dinucleotide phosphate (NADP+) or nicotinamide mononucleotide (NMN+) with an alternative base. 4'-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k(cat)/K(M)=0.9-10 microM(-1)s(-1)) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k(cat)/K(M) values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 microM(-1)s(-1)). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN+, k(cat)/K(M)=0.05-0.4 microM(-1)s(-1). The k(cat)/K(M) values for the exchange reaction with NMN+ were generally similar (0.04-0.12 microM(-1)s(-1)) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k(cat)/K(M) values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.

Pyrrolidinyl pyridone and pyrazinone analogues as potent inhibitors of prolyl oligopeptidase (POP).

We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized within the catalytic site of a human chimeric POP protein which provided a more detailed understanding of how these inhibitors interacted with the key residues within the catalytic pocket.

Inhibition of wild-type and mutant human immunodeficiency virus type 1 proteases by GW0385 and other arylsulfonamides.

The arylsulfonamide derivatives described herein were such potent inhibitors of human immunodeficiency virus type 1 (HIV-1) protease (enzyme, E) that values for the inhibition constants (K(i)) could not be determined by conventional steady-state kinetic techniques (i.e., the minimal enzyme concentration usable for the activity assay was much greater than the value of the dissociation constant). Consequently, two alternative methods were developed for estimation of K(i) values. The first method employed kinetic determinations of values for k(1) and k(-1), from which K(i) was determined (k(-1)/k(1)). The second method was a competitive displacement assay used to determine binding affinities of other inhibitors relative to that of GW0385. In these assays, the inhibitor of unknown affinity was used to displace [(3)H]GW0385 from E.[(3)H]GW0385. From the concentration of E.[(3)H]GW0385 at equilibrium, the concentrations of enzyme-bound and free inhibitors were calculated, and the ratio of the K(i) value of the unknown to that of GW0385 was determined (K(i,unknown)/K(i,GW0385)). The values of k(1) were calculated from data in which changes in the intrinsic protein fluorescence of the enzyme associated with inhibitor binding were directly or indirectly monitored. In the case of saquinavir, the fluorescence changes associated with complex formation were large enough to monitor directly. The value of k(1) for saquinavir was 62 +/- 2 microM(-1) s(-1). In the case of GW0385, the fluorescence changes associated with complex formation were too small to monitor directly. Consequently, the value of k(1) was estimated from a competition experiment in which the effect of GW0385 on the binding of E to saquinavir was determined. The value of k(1) for GW0385 was estimated from these experiments to be 137 +/- 4 microM(-1) s(-1). Because E.[(3)H]GW0385 was stable in the standard buffer at room temperature for greater than 33 days, the value of the first-order rate constant for dissociation of E.[(3)H]GW0385 (k(-1)) could be estimated from the time-course for exchange of E.[(3)H]GW0385 with excess unlabeled GW0385. The value of k(-1) calculated from these data was (2.1 +/- 0.1) x10(-6) s(-1) (t(1/2) = 91 h). The K(i) value of wild-type HIV-1 protease for GW0385, calculated from these values for k(1) and k(-1), was 15 +/- 1 fM. Three multidrug resistant enzymes had K(i) values for GW0385 that were less than 5 pM.

Changes in human immunodeficiency virus type 1 Gag at positions L449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to amprenavir and improved viral fitness in vitro.

Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1'F) and P453L (p1/p6 PP5'L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity approximately 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.

HIV-1 protease: characterization of a catalytically competent enzyme-substrate intermediate.

The steady-state and pre-steady-state kinetic parameters for the interaction of E with the fluorogenic substrate 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO(2))-Gln-Arg-NH(2) were determined in 1.25 M NaCl, 0.1 M MES-TRIS at pH 6.0 at 25 degrees C. At low concentrations of enzyme, the values of the K(m) and k(cat) calculated from steady-state data were 2.1 microM and 7.4 s(-1), respectively. At high concentrations of enzyme, the time-courses of fluorescence enhancement associated with catalysis were very dependent on the excitation wavelength used to monitor the reaction. Because the absorbance spectrum of the substrate overlapped the fluorescence emission spectrum of the enzyme, these abnormalities were attributed to fluorescence energy transfer between the enzyme and the substrate in an enzyme-substrate intermediate. The kinetic data collected with lambda(ex) = 280 nm and lambda(em) > 435 nm were analyzed according to the following mechanism in which EX was the species with enhanced fluorescence relative to substrate or products: [formula see text]. The values of the kinetic parameters with (1)H(2)O as the solvent were K = 13 microM, k(2) = 150 s(-1), k(-2) = 25 s(-1), and k(3) = 11 s(-1). The values of the kinetic parameters with (2)H(2)O as the solvent were K = 13 microM, k(2) = 210 s(-1), k(-2) = 12 s(-1), and k(3) = 4.4 s(-1). These values yielded solvent isotope effects of 2 on k(cat) and 0.9 on k(cat)/K(m). From analysis of the complete time-course of the fluorescence change (lambda(ex) = 280 nm and lambda(em) > 435 nm) during the course of substrate hydrolysis, the intermediate EX was determined to be 6.3-fold more fluorescent than the product, which, in turn, was 4.5-fold more fluorescent than ES or S. Rapid quench experiments with 2 N HCl as the quenching reagent confirmed that EX was a complex between enzyme and substrate. Consequently, the small burst in fluorescence observed when monitoring with lambda(ex) = 340 nm (0.3 product equiv per enzyme equivalent) was attributed to the fluorescence change upon transfer of substrate from an aqueous environment to a nonaqueous environment in the enzyme. These results were consistent with carbon-nitrogen bond cleavage being the major contributor to k(cat).