Identification and characterisation of a new class of highly specific and potent inhibitors of the mitochondrial pyruvate carrier

Two novel thiazolidine compounds, GW604714X and GW450863X, were found to be potent inhibitors of mitochondrial respiration supported by pyruvate but not other substrates. Direct measurement of pyruvate transport into rat liver and yeast mitochondria confirmed that these agents inhibited the mitochondrial pyruvate carrier (MPC) with K(i) values <0.1 muM. Inhibitor titrations of pyruvate-dependent respiration by heart mitochondria gave values (+/-S.E.) for the concentration of inhibitor binding sites (pmol per mg protein) and their K(i) (nM) of 56.0+/-0.9 and 0.057+/-0.010 nM for the more hydrophobic GW604714X; for GW450863X the values were 59.9+/-4.6 and 0.60+/-0.12 nM. [(3)H]-methoxy-GW450863X binding was also used to determine the MPC content of the heart, kidney, liver and brain mitochondria giving values of 56, 40, 26 and 20 pmol per mg protein respectively. Binding to yeast mitochondria was <10% of that in rat liver mitochondria, consistent with the slow rate of pyruvate transport into yeast mitochondria. [(3)H]-methoxy-GW450863X binding was inhibited by GW604714X and by the established MPC inhibitor, UK5099. The absorbance spectra of GW450863X and GW604714X were markedly changed by the addition of beta-mercaptoethanol suggesting that the novel inhibitors, like alpha-cyanocinnamate, possess an activated double bond that attacks a critical cysteine residue on the MPC. However, no labelled protein was detected following SDS-PAGE suggesting that the covalent modification is reversible. GW604714X and GW450863X inhibited l-lactate transport by the plasma membrane monocarboxylate transporter MCT1, but at concentrations more than four orders of magnitude greater than the MPC.     

Analyzing the catalytic mechanism of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

In order to gain insight into the catalytic mechanism of Fe-type nitrile hydratases (NHase), the pH and temperature dependence of the kinetic parameters k cat, K m, and k cat/ K m along with the solvent isotope effect were examined for the Fe-type NHase from Comamonas testosteroni Ni1 ( CtNHase). CtNHase was found to exhibit a bell-shaped curve for plots of relative activity vs pH over pH values 4-10 for the hydration of acrylonitrile and was found to display maximal activity at pH approximately 7.2. Fits of these data provided a p K ES1 value of 6.1 +/- 0.1, a p K ES2 value of 9.1 +/- 0.2 ( k' cat = 10.1 +/- 0.3 s (-1)), a p K E1 value of 6.2 +/- 0.1, and a p K E2 value of 9.2 +/- 0.1 ( k' cat/ K' m of 2.0 +/- 0.2 s (-1) mM (-1)). Proton inventory studies indicate that two protons are transferred in the rate-limiting step of the reaction at pH 7.2. Since CtNHase is stable to 25 degrees C, an Arrhenius plot was constructed by plotting ln( k cat) vs 1/ T, providing an E a of 33.3 +/- 1.5 kJ/mol. Delta H degrees of ionization values were also determined, thus helping to identify the ionizing groups exhibiting the p K ES1 and p K ES2 values. Based on Delta H degrees ion data, p K ES1 is assigned to betaTyr68 while p K ES2 is assigned to betaArg52, betaArg157, or alphaSer116 (NHases are alpha 2beta 2 heterotetramers). Given the strong similarities in the kinetic data obtained for both Co- and Fe-type NHase enzymes, both types of NHase enzymes likely hydrate nitriles in a similar fashion.     

Lateral mobility and specific binding to GABA(A) receptors on hippocampal neurons monitored by fluorescence correlation spectroscopy

The binding behavior of a fluorescently labeled muscimol derivative to the GABA(A) receptor was analyzed at rat hippocampal neurons by fluorescence correlation spectroscopy. After muscimol had been labeled with the fluorophore Alexa Fluor 532, specific binding constants for binding of the dye-labeled ligand (Mu-Alexa) to the GABA(A) receptor were determined. We found a high specific binding affinity of Mu-Alexa with a K(D) value of 3.4 +/- 0.5 nM and a rate constant of ligand-receptor dissociation (k(diss)) of (5.37 +/- 0.95) x 10(-2) s(-1). A rate constant of ligand-receptor association (k(ass)) of (1.57 +/- 0.28) x 10(7) L mol(-1) s(-1) was calculated. The following diffusion coefficients were observed: D(free) = 233 +/- 20 microm(2)/s (n = 66) for free diffusing Mu-Alexa, D(bound1) = 2.8 +/- 0.9 microm(2)/s (n = 64) for the lateral mobility, and D(bound2) = 0.14 +/- 0.05 microm(2)/s (n = 56) for the hindered mobility of the GABA(A) receptor-ligand complex in the cell membrane. Saturation of Mu-Alexa binding was observed at a concentration of 50 nM. A maximum number of binding sites [B(max) = 18.4 +/- -0.4 nM (n = 5)] was found. Similar K(i) values of 4.5 +/- 1.0 nM for nonlabeled muscimol and 8.8 +/- 1.8 nM for Mu-Alexa were found by RRAs using [(3)H]muscimol as a radioligand. A concentration-dependent increase in the level of specific Mu-Alexa binding was demonstrated by the positive cooperative activity of co-incubated midazolam, which was selectively found in GABA(A) receptor-ligand complexes with hindered mobility.     

Filter binding assay for the geldanamycin-heat shock protein 90 interaction

A filter binding assay to measure affinity of [3H-allyl]17-allylamino geldanamycin ([3H]AAG) for the ATP binding site of the N-terminal domain of human Hsp90alpha (hHsp90alpha9-236) was developed. Diethylaminoethyl cellulose or glass fiber filters impregnated with polyethyleneimine were used to capture the [3H]AAG-Hsp90 complex, and conditions which washed >98% of free [3H]AAG from the filters were developed. The complex formed at a rapid rate (k(on)=2.5 x 10(7)Lmol(-1) x s(-1)) and dissociated with a half-life of 2.3 min (k(off)=5 x 10(-3) x s(-1)). hHsp90alpha9-236 bound to [3H]AAG with a K(d) value of 0.4+/-0.1 microM. [3H]AAG had similar affinities for full-length hHsp90alpha and for hHsp90alpha9-236 variants containing biotinylated N-terminal biotinylation signal sequences and N- or C-terminal His(6) tags. Geldanamycin, ADP, ATP, and radicicol-all known to bind to the ATP domain of Hsp90-competed with [3H]AAG for binding to hHsp90alpha9-236, showing K(d) values in good agreement with reported values.     

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.     

Mechanistic studies with 2-C-methyl-D-erythritol 4-phosphate synthase from Escherichia coli

The mechanism of the reaction catalyzed by 2-C-methyl-d-erythritol 4-phosphate (MEP) synthase from Escherichia coli has been studied by steady-state and single-turnover kinetic experiments for the 1-deoxy-d-xylulose 5-phosphoric acid (DXP) analogues, 1,1,1-trifluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(3)-DXP), 1,1-difluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(2)-DXP), 1-fluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF-DXP), and 1,2-dideoxy-d-hexulose 6-phosphate (Et-DXP). CF(3)-DXP, CF(2)-DXP, and Et-DXP were poor inhibitors, most likely because of the increase in steric bulk at C1 of DXP. The three analogues were also poor substrates for the enzyme. In contrast, CF-DXP was a good substrate (k(cat)(CF)(-)(DXP) = 37 +/- 2 s(-)(1), K(m)(CF)(-)(DXP) = 227 +/- 25 microM) for MEP synthase when compared to DXP (k(cat)(DXP) = 29 +/- 1 s(-)(1), K(m)(DXP) = 45 +/- 4 microM). A primary deuterium isotope effect was observed under single-turnover conditions when CF-DXP was incubated with 4S-[(2)H]NADPH ((H)k/(D)k = 1.34 +/-0.01), whereas no isotope effect was observed upon incubation with DXP and 4S-[(2)H]NADPH ((H)k/(D)k = 1.02 +/- 0.02). The reaction did not exhibit burst kinetics for either substrate, indicating that product release is not rate-limiting. These studies suggest that positive charge does not develop at C2 of DXP during catalysis. In addition, the isotope effect with CF-DXP and 4S-[(2)H]NADPH but not DXP indicates that the rearrangement step, which precedes hydride transfer, is rate-limiting for DXP but becomes partially rate-limiting for CF-DXP. Thus, rearrangement appears to be enhanced by substitution of a hydrogen atom in the methyl group of DXP by fluorine. These observations are consistent with a retro-aldol/aldol mechanism for the rearrangement during conversion of DXP to MEP.     

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins.

An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.     

Loop Residues and Catalysis in OMP Synthase

Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.     

Kinetic isotope effect studies on milk xanthine oxidase and on chicken liver xanthine dehydrogenase

The effect of isotopic substitution of the 8-H of xanthine (with 2H and 3H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in H/T(V/K) values was observed between xanthine oxidase (3.59 +/- 0.1) and xanthine dehydrogenase (3.60 +/- 0.09). Xanthine dehydrogenase exhibited a larger T/D(V/K) value (0.616 +/- 0.028) than that observed for xanthine oxidase (0.551 +/- 0.016). Observed H/T(V/K) values for either enzyme are less than those H/T(V/K) values calculated with D/T(V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8-3H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (Cr) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect (Dk) for xanthine oxidation is calculated to be 7.4 +/- 0.7 for xanthine oxidase and 4.2 +/- 0.2 for xanthine dehydrogenase. By use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be approximately 75-fold faster than kcat for xanthine oxidase and approximately 10-fold faster than kcat for xanthine dehydrogenase. This calculated rate is consistent with data obtained by rapid-quench experiments with XO. A stoichiometry of 1.0 +/- 0.3 mol of uric acid/mol of functional enzyme is formed within the mixing time of the instrument (5-10 ms). The kinetic isotope effect data also permitted the calculation of the Kd values [Klinman, J. P., & Mathews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] for substrate dissociation, including all reversible steps prior to C-H bond cleavage. Values calculated for each enzyme (Kd = 120 microM) were found to be identical within experimental uncertainty.     

Importance of product/reactant equilibration in the kinetics of the phosphoglucose isomerization reaction by differential stopped flow microcalorimetry

The kinetics for the isomerization of fructose-6-phosphate to glucose-6-phosphate (F6P --> G6P) by baker's yeast phosphoglucose isomerase (PGI) with regard to k(cat) and K(m) were determined from analysis of differential stopped flow microcalorimeter measurements using the integrated form of the Michaelis-Menten rate equation. Values for K(m) (F6P --> G6P) that were determined at pH 8.0 and ionic strength 0.1M at 293.4, 298.4, 303.4, and 311.5K exhibited a linear dependence on the substrate concentration at each temperature because of the substrate-product equilibrium. The minimum values for K(m) ranged from 2.62+/-0.55 mM at 293.4K to 7.8+/-4.8mM at 311.5K and were the same as the minimum values for the reverse reaction (G6P --> F6P) at 293.4 K and 298.4 K. Minimum values for k(cat) increased with temperature, from 2.78+/-0.34s(-1) at 293.4K to 11.4+/-1.0s(-1) at 311.5K, and for the reverse reaction, G6P --> F6P, from 0.852+/-0.086 s(-1) at 293.4K to 1.46+/-0.06s(-1) at 298.4K. The enzyme efficiency at 311.5K is close to the collision rate for a diffusion-controlled process in solution. The [F6P]/[G6P] equilibrium constants were determined from comparison of the values of k(cat) in both directions and were 0.307+/-0.053 at 293.4K and 0.395+/-0.033 at 298.4K. The heats of reaction in the F6P --> G6P direction increased from -8.96+/-0.26 kJmol(-1) at 311.5K to -8.27+/-0.40 kJmol(-1) at 293.4K, a value in fair agreement with 7.01+/-0.32 kJmol(-1) in the opposite G6P --> F6P direction.     

Stopped-flow kinetic study of vitamin E regeneration reaction with biological hydroquinones (reduced forms of ubiquinone, vitamin K, and tocopherolquinone) in solution.

A kinetic study of the regeneration reaction of vitamin E (tocopherol) with eight biological hydroquinones (HQs) (ubiquinol-10 (Q10H2 1); ubiquinol-0 (Q0H2 2); vitamin K1 HQ (VK1H2 3); vitamin K3 HQ (VK3H2 4); alpha-, beta-, and gamma-tocopherol-HQs (alpha-, beta-, and gamma-TQH2 5-7); and 2,3,5-trimethyl-1,4-HQ (TMQH2 8)) in solution was performed. The second-order rate constants (k4) for the reaction of HQs 1-8 with alpha-tocopheroxyl and 5,7-diisopropyltocopheroxyl radicals in ethanol, benzene, and isopropyl alcohol/water (5:1, v/v) solutions were measured with a stopped-flow spectrophotometer. The order of magnitude of k4 values obtained for HQs is VK1H2 > VK3H2 > alpha-TQH2 > beta-TQH2 approximately gamma-TQH2 approximately TMQH2 > Q10H2 > Q0H2, being independent of the kinds of tocopheroxyl radicals and the polarity of the solvents. The log of the k4 values obtained for HQs was found to correlate with their peak oxidation potentials. Comparing the k2 value (2.68 x 10(6) M-1 s-1 obtained for the reaction of alpha-tocopheroxyl with vitamin C (sodium ascorbate) with those (k4 = 2.54 x 10(5) and 8.15 x 10(5) M-1 s-1) obtained for the reaction of alpha-tocopheroxyl with Q10H2 and alpha-TQH2 in isopropyl alcohol/water mixtures, the former is approximately 11 and 3 times as reactive as the latter, respectively. On the other hand, the k2 value obtained for sodium ascorbate is smaller than the k4 values obtained for VK1H2 and VK3H2. These results suggest that mixtures of vitamin E and these HQs (as well as those of vitamins E and C) may function synergistically as antioxidants in various tissues and mitochondria.     

Changes in pH associated with clotting of fibrinogen. Kinetic studies of the pH shift and correlation of the pH change with the release of fibrinopeptides and the ensuing polymerization

The effect of the initial pH and the concentrations of thrombin, fibrinogen, and Ca2+ upon the rate of pH change associated with clotting of bovine fibrinogen by human thrombin was investigated at pH 6.80, 7.80, and 8.80, 0.3 ionic strength, 25 degrees C, and 19.5 mg/mL final fibrinogen concentration. At pH 6.80 and 7.80, the reaction was first order, with rate constant k1. At pH 8.80, a first-order reaction of the release of H+ (k1) was followed by a partial rebinding of these in a reaction consecutive to the first one (k2). At each of the above pH values, k1 was proportional to thrombin concentration in the 0.05-3.0 min-1 range investigated. The k1 constants were 0.111 +/- 0.001, 0.250 +/- 0.005, and 0.190 +/- 0.002 min-1 (NIH thrombin units)-1 mL-1 at pH 6.80, 7.80, and 8.80, respectively. Plots of log rate vs log thrombin concentration of these data were linear with slopes close to 1 at all three pH values. The rate of the second reaction (k2) was independent of both the thrombin and the initial fibrinogen concentration. The pH dependence of k1 exhibited a bell-shaped curve that could be resolved into the effect of one group with a pK of 7.27 that increased the rate and another with a pK of 9.22 that decreased the rate. With constant thrombin concentration but varying fibrinogen concentration, plots of 1/k1 vs [fibrinogen] were linear, but the lines did not pass through the origin. From the slope and intercept, kcat and KM of the Michaelis-Menten equation could be calculated. The same parameters were obtained also from initial velocity vs [fibrinogen] plots. Values of kcat were consistent and accurate; those of KM were more scattered. KM was (22.4-34.2) X 10(-6) M at pH 6.80 and approximately 7 X 10(-6) M in the pH 7.26-8.80 range. The latter value, pertaining to the release of H+ ions, is in agreement with values in the literature for KM of the release of fibrinopeptide A by thrombin in the 7.4-8.0 pH range. The value of kcat s-1 (unit of thrombin)-1 mL-1 increases from 1.2 X 10(-10) s-1 unit of thrombin-1 mL-1 at pH 6.80 to 2.46 X 10(-10) at pH 7.80 and then decreases to 2.01 X 10(-10) 10(-1) (units of thrombin)-1 mL-1 at pH 8.80. The kcat values are significantly lower than those in the literature for the release of fibrinopeptide A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple-ligand binding in CYP2A6: probing mechanisms of cytochrome P450 cooperativity by assessing substrate dynamics

The contribution of ligand dynamics to CYP allosterism has not been considered in detail. On the basis of a previous study, we hypothesized that CYP2A6 and CYP2E1 accommodate multiple xylene ligands. As a result, the intramolecular ( k H/ k D) obs values observed for some xylene isomers are expected to be dependent on ligand concentration with contributions from [CYP.xylene] and [CYP.xylene.xylene], etc. To explore this possibility and the utility of kinetic isotope effects in characterizing allosteric CYP behavior, steady state kinetics, product ratios, and ( k H/ k D) obs values for CYP2E1 and CYP2A6 oxidation of m-xylene-alpha- (2)H 3 and p-xylene-alpha- (2)H 3 were determined. Evidence is presented that CYP2A6 accommodates multiple ligands and that intramolecular isotope effect experiments can provide insight into the mechanisms of multiple-ligand binding. CYP2A6 exhibited cooperative kinetics for m-xylene-alpha- (2)H 3 oxidation and a concentration-dependent decrease in the m-methylbenzylalcohol:2,4-dimethylphenol product ratio (9.8 +/- 0.1 and 4.8 +/- 0.3 at 2.5 microM and 1 mM, respectively). Heterotropic effects were observed as well, as incubations containing both 15 microM m-xylene-alpha- (2)H 3 and 200 microM p-xylene resulted in further reduction of the product ratio (2.4 +/- 0.2). When p-xylene (60 microM) was replaced with deuterium-labeled d 6- p-xylene (60 microM), an intermolecular competitive inverse isotope effect on 2,4-dimethylphenol formation [( k H/ k D) obs = 0.49] was observed, indicating that p-xylene exerts heterotropic effects by residing in the active site simultaneously with m-xylene. The data indicate that there is a concentration-dependent decrease in the reorientation rate of m-xylene, as no increase in ( k H/ k D) obs was observed in the presence of an increased level of metabolic switching. That is, the accommodation of a second xylene molecule in the active site leads to a decrease in substrate dynamics.     

Protein kinase A: purification and characterization of the enzyme from two cold-hardy goldenrod gall insects

The catalytic subunit of protein kinase A (PKAc) was purified to apparent homogeneity from two species of cold-hardy goldenrod gall insects, Epiblema scudderiana and Eurosta solidaginis. Final specific activity for both enzymes was approximately 74.5 nmol of phosphate transferred per minute per milligram protein. Molecular weights were 41 and 40 kDa for E. scudderiana and E. solidaginis PKAc, respectively. K(m) values at 24 degrees C for the artificial substrate, Kemptide, were 38.1+/-4.9 and 3.67+/-0.11 microM for E. scudderiana and E. solidaginis PKAc, respectively, whereas K(m) Mg-ATP values were 61.1+/-6.9 and 30.7+/-4.1 microM. Assay at 4 degrees C lowered the K(m) for Kemptide of E. scudderiana PKAc by 55% and addition of 1M glycerol further lowered the K(m). Low assay temperature also enhanced holoenzyme dissociation in both species with the K(a) value for cyclic 3'5'-monophosphate at 4 degrees C lowered to just 13-18% of the value at 24 degrees C. Low temperature did not affect affinity for Mg-ATP or inhibition by PKA inhibitors (PKAi, H7, H89) but increased inhibition by some salts. PKAc from both species showed a break in the Arrhenius relationship at approximately 10 degrees C which suggests a conformational change at low temperature; activation energies (E(a)) were 2.2-3 fold higher for the lower (<10 degrees C) versus higher (>10 degrees C) range. Addition of naturally occurring polyols, 1M glycerol or 0.4M sorbitol, affected E(a) in some cases. Temperature dependent regulation of holoenzyme dissociation and PKAc kinetic properties may have an role in regulating the enzymes involved in polyol synthesis in cold-hardy insects.     

Structural and mechanistic insight into the inhibition of aspartic proteases by a slow-tight binding inhibitor from an extremophilic Bacillus sp.: correlation of the kinetic parameters with the inhibitor induced conformational changes

We present here the first report of a hydrophilic peptidic inhibitor, ATBI, from an extremophilic Bacillus sp. exhibiting a two-step inhibition mechanism against the aspartic proteases, pepsin and F-prot from Aspergillus saitoi. Kinetic analysis shows that these proteases are competitively inhibited by ATBI. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I right arrow over left arrow (k(3), k(4)) EI right arrow over left arrow (k(5), k(6)) EI. The K(i) values for the first reversible complex (EI) of ATBI with pepsin and F-prot were (17 +/- 0.5) x 10(-9) M and (3.2 +/- 0.6) x 10(-6) M, whereas the overall inhibition constant K(i) values were (55 +/- 0.5) x 10(-12) M and (5.2 +/- 0.6) x 10(-8) M, respectively. The rate constant k(5) revealed a faster isomerization of EI for F-prot [(2.3 +/- 0.4) x 10(-3) s(-1)] than pepsin [(7.7 +/- 0.3) x 10(-4) s(-1)]. However, ATBI dissociated from the tight enzyme-inhibitor complex (EI) of F-prot faster [(3.8 +/- 0.5) x 10(-5) s(-1)] than pepsin [(2.5 +/- 0.4) x 10(-6) s(-1)]. Comparative analysis of the kinetic parameters with pepstatin, the known inhibitor of pepsin, revealed a higher value of k(5)/k(6) for ATBI. The binding of the inhibitor with the aspartic proteases and the subsequent conformational changes induced were monitored by exploiting the intrinsic tryptophanyl fluorescence. The rate constants derived from the fluorescence data were in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI. Chemical modification of the Asp or Glu by WRK and Lys residues by TNBS abolished the antiproteolytic activity and revealed the involvement of two carboxyl groups and one amine group of ATBI in the enzymatic inactivation.     

Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus

The catalytic mechanism underlying the aminopeptidase from Streptomyces griseus (SGAP) was investigated. pH-dependent activity profiles revealed the enthalpy of ionization for the hydrolysis of leucine-para-nitroanilide by SGAP. The value obtained (30 +/- 5 kJ.mol(-1)) is typical of a zinc-bound water molecule, suggesting that the zinc-bound water/hydroxide molecule acts as the reaction nucleophile. Fluoride was found to act as a pure noncompetitive inhibitor of SGAP at pH values of 5.9-8 with a K(i) of 11.4 mM at pH 8.0, indicating that the fluoride ion interacts equally with the free enzyme as with the enzyme-substrate complex. pH-dependent pK(i) experiments resulted in a pK(a) value of 7.0, suggesting a single deprotonation step of the catalytic water molecule to an hydroxide ion. The number of proton transfers during the catalytic pathway was determined by monitoring the solvent isotope effect on SGAP and its general acid-base mutant SGAP(E131D) at different pHs. The results indicate that a single proton transfer is involved in catalysis at pH 8.0, whereas two proton transfers are implicated at pH 6.5. The role of Glu131 in binding and catalysis was assessed by determining the catalytic constants (K(m), k(cat)) over a temperature range of 293-329 degrees K for both SGAP and the E131D mutant. For the binding step, the measured and calculated thermodynamic parameters for the reaction (free energy, enthalpy and entropy) for both SGAP and the E131D mutant were similar. By contrast, the E131D point mutation resulted in a four orders of magnitude decrease in k(cat), corresponding to an increase of 9 kJ.mol(-1) in the activation energy for the E131D mutant, emphasizing the crucial role of Glu131 in catalysis.     

Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.

The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western blot analysis, respectively. As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts. In the presence of 5 microM phosphate, Na+ promotes phosphate uptake into yeast cells expressing the wild-type NaPi-2 with a K(0.5) of 5.6 +/- 1.1 mM. The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min). Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min under the same conditions. The K(0.5) for the stimulation of phosphate uptake by Na+ is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly different from the value obtained with cells expressing the wild-type cotransporter. The reduced level of accumulation of phosphate in yeast cells expressing the R215E mutant is probably due to a reduction of the first-order rate constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate uptake into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefore about 4-fold lower. In comparison, the rate for phosphate uptake into nontransformed cells (k) is 0.0075 min(-1). Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promoted by extracellular phosphate with a K(0.5) of 45 +/- 4 microM. A phosphate-dependent phosphate accumulation is also observed with cells expressing the R215E mutant, but the K(0.5) is twice as high (86 +/- 5 microM) as that obtained with the wild-type cotransporter. We conclude that the yeast expression system is a useful tool for the investigation of structure-function relationships of the renal sodium/phosphate cotransporter and that (215)R, although not involved in Na+ recognition, is a part of the structure involved in phosphate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter.     

Enzymatic characterization and inhibitor discovery of a new cystathionine {gamma}-synthase from Helicobacter pylori

Cystathionine gamma-synthase (CGS) catalyses the first step of the transsulfuration pathway that converts l-cysteine to l-homocysteine in bacteria, whereas this pathway is absent in human. In this report, we identified a new metB gene from Helicobacter pylori strain SS1, and the recombinant H. pylori Cystathionine gamma-synthase (HpCGS) was successfully cloned, expressed and purified in Escherichia coli system. Enzymatic study of HpCGS indicated that the K(m) and k(cat)/K(m) values against the substrate O-succinyl-l-homoserine (l-OSHS) were 3.02 mM and 98.7 M(-)(1)s(-)(1), respectively. Moreover, four natural products (alpha-lapachone, 9-hydroxy-alpha-lapachone, Paulownin and Yangambin, Fig. 1) were discovered to demonstrate inhibitory activities against HpCGS with IC(50) values of 11 +/- 3, 9 +/- 1, 19 +/- 2 and 27 +/- 6 microM, respectively. All these four inhibitors prevent the binding of l-OSHS to HpCGS in a non-competitive fashion. In vitro antibacterial assays further indicated that these four discovered compounds could highly inhibit the growth of H. pylori and exhibited strong inhibitory specificity against H. pylori related to E. coli.     

Unraveling the catalytic mechanism of nitrile hydratases

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.