Quantitative structure toxicity relationships for catechols in isolated rat hepatocytes

One- and two-parameter quantitative structure toxicity relationship (QSTR) equations were obtained to describe the cytotoxicity of isolated rat hepatocytes induced by 23 catechols in which LD(50) represents the catechol concentration required to induce 50% cytotoxicity in 2 h. A QSTR equation logLD(50) (microM = - 0.464(+/-0.065) log P + 3.724(+/-0.114) (n = 20, r(2) = 0.740, s(y,x) = 0.372, P < 1 x 10(-6), outliers: 4-methoxycatechol, 3-methoxycatechol, L-dopa) was derived where logP represents octanol/water partitioning. Outliers were determined by adopting a statistical method to standardize the identification of outliers. When pK(a1), the first ionization constant, was considered as a contributing parameter a two-parameter QSTR equation was derived: logLD(50) (microM = - 0.343(+/-0.058) log P - 0.116(+/-0.041) pK(a1)+4.389 (+/-0.315) (n = 22, r(2) = 0.738, s(y,x) = 0.375, P < 0.01, outlier: 4-methoxycatechol). Replacing logP with logD(7.4), the partition coefficient at pH 7.4, improved the first correlation by limiting the outlier to 4-methoxycatechol: logLD(50) (microM)=-0.252(+/-0.039) logD(7.4)+3.168(+/-0.090) (n = 22, r(2) = 0.671, s(y,x) = 0.420, P < 1 x 10(-5). In this study, 4-methoxycatechol (readily autooxidizable) was found to be an outlier for all QSTR equations derived. These findings point to lipophilicity and pK(a1) as two important characteristics of catechols that can be used to predict their cytotoxicity towards isolated rat hepatocytes. The catechols with the higher lipophilicity/distribution coefficient, the lower degree of ionization and the higher pK(a(catechol)) were more toxic towards hepatocytes than the other catechols.     

Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6- fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.     

Evaluation of tetraether lipid-based liposomal carriers for encapsulation and retention of nucleoside-based drugs

Although liposomal nanoparticles are one of the most versatile class of drug delivery systems, stable liposomal formulation of small neutral drug molecules still constitutes a challenge due to the low drug retention of current lipid membrane technologies. In this study, we evaluate the encapsulation and retention of seven nucleoside analog-based drugs in liposomes made of archaea-inspired tetraether lipids, which are known to enhance packing and membrane robustness compared to conventional bilayer-forming lipids. Liposomes comprised of the pure tetraether lipid generally showed improved retention of drugs (up to 4-fold) compared with liposomes made from a commercially available diacyl lipid. Interestingly, we did not find a significant correlation between the liposomal leakage rates of the molecules with typical parameters used to assess lipophilicity of drugs (such logD or topological polar surface area), suggesting that specific structural elements of the drug molecules can have a dominant effect on leakage from liposomes over general lipophilic character.     

Acute rosiglitazone treatment is cardioprotective against ischemia-reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice

Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, has been demonstrated to possess cardioprotective properties during ischemia-reperfusion. However, this notion remains controversial as recent evidence has suggested an increased risk in cardiac events associated with long-term use of RGZ in patients with type 2 diabetes. In this study, we tested the hypothesis that acute RGZ treatment is beneficial during I/R by modulating cardioprotective signaling pathways in a nondiabetic mouse model. RGZ (1 μg/g) was injected intravenously via the tail vein 5 min before reperfusion. Myocardial infarction was significantly reduced in mice treated with RGZ compared with vehicle controls (8.7% ± 1.1% vs. 20.2% ± 2.5%, P < 0.05). Moreover, isolated hearts were subjected to 20 min of global, no-flow ischemia in an ex vivo heart perfusion system. Postischemic recovery was significantly improved with RGZ treatment administered at the onset of reperfusion compared with vehicle (P < 0.001). Immunoblot analysis data revealed that the levels of both phospho-AMP-activated protein kinase (Thr(172)) and phospho-Akt (Ser(473)) were significantly upregulated when RGZ was administered 5 min before reperfusion compared with vehicle. On the other hand, inflammatory signaling [phospho-JNK (Thr(183)/Tyr(185))] was significantly downregulated as a result of RGZ treatment compared with vehicle (P < 0.05). Intriguingly, pretreatment with the selective PPAR-γ inhibitor GW-9662 (1 μg/g iv) 10 min before reperfusion significantly attenuated these beneficial effects of RGZ on the ischemic heart. Taken together, acute treatment with RGZ can reduce ischemic injury in a nondiabetic mouse heart via modulation of AMP-activated protein kinase, Akt, and JNK signaling pathways, which is dependent on PPAR-γ activation.     

Effects of Ca(2+)-activated calmodulin on neuronal nitric oxide synthase reductase activity and binding of substrates: pH dependence of kinetic parameters.

The pH dependence of basal and calmodulin- (CaM-) stimulated neuronal nitric oxide synthase (nNOS) reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) was investigated. The wave-shaped log V versus pH profile revealed that optimal DCIP reduction occurred when a group, pK(a) of 7.6-7.8, was ionized. The (V/K)(NADPH) and (V/K)(DCIP) versus pH profiles increased with the protonation of a group with a pK(a) of 6.5 or 5.9 and the ionization of two groups with the same pK(a) of 7.5 or 7.0, respectively. (V/K)(DCIP) decreased with the ionization of a group, pK(a) of 9.0. Similar V, (V/K)(NADPH), and (V/K)(DCIP) versus pH profiles for DCIP reduction were obtained with and without CaM, indicating that CaM does not influence ionizable groups involved in catalysis or substrate binding. In contrast, CaM affected the pH dependence of cytochrome c(3+) reduction. The wave-shaped log V versus pH profile for basal cytochrome c(3+) reduction revealed that ionization of a group, pK(a) of 8.6, increased catalysis. Log V for CaM-stimulated cytochrome c(3+) reduction displayed a bell-shaped pH dependence with the protonation of a group with a pK(a) of 6.4 and the ionization of a group with a pK(a) of 9.3, resulting in a loss of activity. The log(V/K)(cytc) versus pH profiles with and without CaM were bell-shaped with the ionization of a group at pK(a) of 7.1 or 7.6 (CaM) or pK(a) of 9.4 or 9.6 (CaM), increasing and decreasing (V/K)(cytc). These results suggest that CaM may change the nature of the rate-limiting catalytic steps or ionizable groups involved in cytochrome c(3+) reduction.     

Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential.

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane surface pH compared to the bulk pH, increasing K(eq) and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-)(1)) of the MI-MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H(+)/MII. We find that the apparent fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent with titration of a single H(+) binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.     

Rosiglitazone treatment restores renal responsiveness to atrial natriuretic peptide in rats with congestive heart failure

The thiazolidinedione (TZD) class of Peroxisome proliferator‐activated receptor gamma agonists has restricted clinical use for diabetes mellitus due to fluid retention and potential cardiovascular risks. These side effects are attributed in part to direct salt‐retaining effect of TZDs at the renal collecting duct. A recent study from our group revealed that prolonged rosiglitazone (RGZ) treatment caused no Na+/H₂O retention or up‐regulation of Na⁺ transport‐linked channels/transporters in experimental congestive heart failure (CHF) induced by surgical aorto‐caval fistula (ACF). The present study examines the effects of RGZ on renal and cardiac responses to atrial natriuretic peptide (ANP), Acetylcholine (Ach) and S‐Nitroso‐N‐acetylpenicillamine (SNAP‐NO donor). Furthermore, we assessed the impact of RGZ on gene expression related to the ANP signalling pathway in animals with ACF. Rats subjected to ACF (or sham) were treated with either RGZ (30 mg/kg/day) or vehicle for 4 weeks. Cardiac chambers pressures and volumes were assessed invasively via Miller catheter. Kidney excretory and renal hemodynamic in response to ANP, Ach and SNAP were examined. Renal clearance along with cyclic guanosine monophosphate (cGMP), gene expression of renal CHF‐related genes and ANP signalling in the kidney were determined. RGZ‐treated CHF rats exhibited significant improvement in the natriuretic responses to ANP infusion. This ‘sensitization’ to ANP was not associated with increases in neither urinary cGMP nor in vitro cGMP production. However, RGZ caused down‐regulation of several genes in the renal cortex (Ace, Nos3 and Npr1) and up‐regulation of ACE2, Agtrla, Mme and Cftr along down‐regulation of Avpr2, Npr1,2, Nos3 and Pde3 in the medulla. In conclusion, CHF+RGZ rats exhibited significant enhancement in the natriuretic responses to ANP infusion, which are known to be blunted in CHF. This ‘sensitization’ to ANP is independent of cGMP signalling, yet may involve post‐cGMP signalling target genes such as ACE2, CFTR and V2 receptor. The possibility that TZD treatment in uncomplicated CHF may be less detrimental than thought before deserves additional investigations.     

Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH

Potentiators are molecules that increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Some potentiators can also inhibit CFTR at higher concentrations. The activating binding site is thought to be located at the interface of the dimer formed by the two nucleotide-binding domains. We have hypothesized that if binding of potentiators involves titratable residues forming salt bridges, then modifications of cytosolic pH (pH(i)) would alter the binding affinity. Here, we analyzed the effect of pH(i) on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pH(i) does modify CFTR maximum current (I(m)) and half-activation concentration (K(d)): I(m) = 127.7, 185.5, and 231.8 μA/cm(2) and K(d) = 32.7, 56.6 and 71.9 μm at pH 6, 7.35, and 8, respectively. We also found that the genistein apparent dissociation constant for activation (K(a)) increased at alkaline pH(i), near cysteine pK (K(a) = 1.83, 1.81 and 4.99 μm at pH(i) 6, 7.35, and 8, respectively), suggesting the involvement of cysteines in the binding site. Mutations of cysteine residues predicted to be within (Cys-491) or outside (Cys-1344) the potentiator-binding site showed that Cys-491 is responsible for the sensitivity of potentiator binding to alkaline pH(i). Effects of pH(i) on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pH(i) on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.     

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of electrostatic binding sites of extracellular polymers by linear programming analysis of titration data

Electrostatic binding sites of extracellular polymeric substances (EPS) were characterized from titration data using linear programming analysis. Test results for three synthetic solutions of given solutes comprising amino, carboxyl, and phenolic groups indicated that this method was able to identify the electrostatic binding sites. For the six sites with pK(a) between 3 and 10, the estimated pK(a) deviated 0.11 +/- 0.09 from the theoretical values, and the estimated concentrations deviated 3.0% +/- 0.9% from the actual concentrations. Two EPS samples were then extracted from a hydrogen-producing sludge (HPS) and a sulfate-reducing biofilm (SRB). Analysis of charge excess data in titration from pH 3 to 11 indicated that the EPS of HPS comprised of five electrostatic binding sites with pK(a) ranging from 3 to 11. The pK(a) values of these binding sites and the possible corresponding functional groups were pK(a) 4.8 (carboxyl), pK(a) 6.0 (carboxyl/phosphoric), pK(a) 7.0 (phosphoric), pK(a) 9.8 (amine/phenolic), and pK(a) 11.0 (hydroxyl). EPS of the SRB comprised five of similar binding sites (with corresponding pK(a) values of 4.4, 6.0, 7.4, 9.4, and 11.0), plus one extra site at pK(a) 8.2, which was likely corresponding to the sulfhydryl group. The total electrostatic binding site concentration of EPS extracted from HPS were 10.88 mmol/g-EPS, of which the highest concentration was from the site of pK(a) 11.0. The corresponding values for the EPS extracted from SRB were 16.44 mmol/g-EPS and pK(a) 4.4. The total concentrations of electrostatic binding sites found in this study were 20- to 30-fold of those reported for bacterial cell surface, implying that EPS might be more crucial in biosorption of metals than bacterial cell surface in wastewater treatment and in bioremediation.     

Synthesis, biological activity, QSAR and QSPR study of 2-aminobenzimidazole derivatives as potent H3-antagonists

We report the design, synthesis, QSPR and QSAR of a new class of H(3)-antagonists, having a 2-aminobenzimidazole moiety connected to the 4(5) position of an imidazole ring through di- or tri-methylene chains. Eleven substituents, selected by experimental design to obtain broad and non-correlated variation in their lipophilic, electronic and steric properties, were introduced at the 5(6) position of the benzimidazole nucleus. The compounds were tested for their H(3)-receptor affinity, by displacement of [(3)H]-(R)-alpha-methylhistamine ([(3)H]-RAMHA) binding to rat brain membranes (pK(i)), for intrinsic activity, evaluating their effect on [(35)S]GTPgammaS binding to rat brain membranes, and for H(3)-antagonist potency, on electrically stimulated guinea-pig ileum (pK(B)). The pK(i) values of the derivatives with longer chain (5a-k) ranged over 2 orders of magnitude, with the 5(6)-methoxy derivative 5d endowed with sub-nanomolar affinity (pK(i)=9.37). The series having two methylene groups in the chain spacer (4a-k), showing a small variation in affinity, revealed to be somewhat insensitive to ring substitution. Lipophilicity (log P) and basicity (pK(a)) of the newly synthesized compounds were measured and related to receptor affinity in a QSAR study. Multiple regression analysis (MRA) showed an approximate parabolic dependence of pK(i) on log P, while an additional electronic effect of the substituents on benzimidazole tautomerism is suspected.     

Binding of substrate analogs to hen egg-white lysozyme with an ester linkage between Glu 35 and Trp 108.

The interactions of the substrate analogs beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which an ester linkage had been formed between Glu 35 and Trp 108 (108 ester lysozyme), were studied by the circular dichroic and fluorescence techniques, and were compared with those for intact lysozyme. The binding constants of beta-methyl-GlcNAc and (GlcNAc)2 to 108 ester lysozyme were essentially the same as those for intact lysozyme in the pH range of 1 to 5. Above pH 5, the binding constants of these saccharides to 108 ester lysozyme did not change with pH, while the binding constants to intact lysozyme decreased. This indicates that Glu 35 (pK 6.0 in intact lysozyme) participates in the binding of these saccharides. The extent and direction of the pK shifts of Asp 52 (pK 3.5), Asp 48 (pK 4.4), and Asp 66 (pK 1.3) observed when beta-methyl-GlcNAc is bound to 108 ester lysozyme were the same as those for intact lysozyme. The participation of Asp 101 and Asp 66 in the binding of (GlcNAc)2 to 108 ester lysozyme was also the same as that for intact lysozyme. These findings indicate that the conformations of subsites B and C are not changed by the formation of the ester linkage. On the other hand, the binding constants of (GlcNAc)3 to 108 ester lysozyme were higher than those for intact lysozyme at all pH values studied. This result is interpreted in terms of an increase in the affinity for a GlcNAc residue of subsite D, which is situated near the esterified Glu 35.     

Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity

Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The K(m) values for the catecholic substrate (K(mA)) and O(2) (K(mO2)), and catalytic constants (k(cat)) were kinetically determined for eight C3/C4-substituted catechols at 25 degrees C and pH 6.5 or 7.5. The first pK(a) values (pK(1)) were determined for eleven catechols (pK(1) = 7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of k(cat)/K(mA) (substrate specificity constant) exhibited a good linear correlation with pK(1), with the exception of those for 4-halocatechols. The logarithm of k(cat)/K(mO2) showed a good linear correlation with pK(1), with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O(2) binding, and the activation of the bound O(2) are all sensitive to electronic effects of the substituents. However, k(cat) did not correlate significantly with pK(1). The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.     

Synthesis, characterization, and DNA adsorption studies of ampholytic model conetworks based on cross-linked star copolymers

Five model conetworks based on cross-linked star ampholytic copolymers were synthesized by group transfer polymerization. The ampholytic copolymers were based on two hydrophilic monomers: the positively ionizable 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the negatively ionizable methacrylic acid (MAA). Ethylene glycol dimethacrylate was used as the cross-linker. These five ampholytic model conetworks were isomers based on equimolar DMAEMA-MAA copolymer stars of different architectures: heteroarm (two), star block (two), and statistical. The two networks based on the homopolymer stars were also synthesized. The MAA units were introduced via the polymerization of tetrahydropyranyl methacrylate and the acid hydrolysis of the latter after network formation. All the precursors to the (co)networks were characterized in terms of their molecular weights using gel permeation chromatography (GPC). The mass of the extractables from the (co)networks was measured and characterized in terms of molecular weight and composition using GPC and proton nuclear magnetic resonance (1H NMR) spectroscopy, respectively. The degrees of swelling (DS) of all the ampholytic conetworks were measured as a function of pH and were found to present a minimum at a pH value which was taken as the isoelectric point, pI. The DS and the pI values did not present a dependence on conetwork architecture. Finally, DNA adsorption studies onto the ampholyte conetworks indicated that DNA binding was governed by electrostatics.     

Stimulation of ENaC Activity by Rosiglitazone is PPARγ-Dependent and Correlates with SGK1 Expression Increase

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type 2 diabetes. TZD treatment induces side effects such as peripheral fluid retention, often leading to discontinuation of therapy. Previous studies have shown that PPARγ activation by TZD enhances the expression or function of the epithelial sodium channel (ENaC) through different mechanisms. However, the effect of TZDs on ENaC activity is not clearly understood. Here, we show that treating Xenopus laevis oocytes expressing ENaC and PPARγ with the TZD rosiglitazone (RGZ) produced a twofold increase of amiloride-sensitive sodium current (Iam), as measured by two-electrode voltage clamp. RGZ-induced ENaC activation was PPARγ-dependent since the PPARγ antagonist GW9662 blocked the activation. The RGZ-induced Iam increase was not mediated through direct serum- and glucocorticoid-regulated kinase (SGK1)-dependent phosphorylation of serine residue 594 on the human ENaC α-subunit but by the diminution of ENaC ubiquitination through the SGK1/Nedd4-2 pathway. In accordance, RGZ increased the activity of ENaC by enhancing its cell surface expression, most probably indirectly mediated through the increase of SGK1 expression.     

Solvent isotope effects and the pH dependence of laccase activity under steady-state conditions

Solvent isotope effects and the pH dependence of laccase catalysis under steady-state conditions were examined with a rapid reductant to assess the potential roles of protein protic groups and the catalytic mechanism. The pH dependence of both reductant-dependent and reductant-independent steps showed bell-shaped profiles implicating at least two protic groups in each case. The apparent pKa values were: for the reductant-independent step(s), pK alpha 1 = 8.98 +/- 0.02 and pK alpha 2 = 5.91 +/- 0.03; for the reductant-dependent step(s), pK' alpha 1 = 7.55 +/- 0.12, pK' alpha 2 = 8.40 +/- 0.23. No solvent isotope effect on reductant-dependent steps was detected other than a standard shift effect. However, a significant solvent isotope effect on a reductant-independent step(s) was observed; kH/kD = 2.12 at the pH optimum of 7.5. The concentration dependence of the D2O effect indicated that a single proton was involved. Simulations of the p(H,D) data suggested that the solvent isotope effect was associated with the protein protic group required in its undissociated form (pK alpha 2). The pH effects on reductant-dependent steps are apparently associated with reductant-dependent steps that occur between O2 binding and water formation in the catalytic reaction sequence.     

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.     

Interactions of alpha- and beta-N-acetyl-D-glucosamines with hen and turkey lysozymes.

The binding constants of alpha- and beta-GlcNAc to hen and turkey lysozymes [EC 3.2.1.17] were determined at various pH's using the method proposed by Ikeda and Hamaguchi (1975) J. Biochem. 77, 1-16). The pH dependence of the binding of beta-GlcNAc to hen lysozyme was essentially the same as that for turkey lysozyme. The pH dependence curves of the binding constants of beta-GlcNAc to hen and turkey lysozymes were interpreted in terms of the participation of Glu 35 (pK 6.0), Asp 52 (pK 3.5), Asp 48 (pK 4.5), and Asp 66 (pK 1.5). The binding constants of alpha-GlcNAc to hen and turkey lysozymes were the same below pH 3.5 but were different above this pH. The main participant residues in the binding of alpha-GlcNAc were Glu 35, Asp 48, and Asp 66 for hen lysozyme and Glu 35 and Asp 66 for turkey lysozyme. The results obtained here were well explained by the following assumptions: (1) above about pH 4, alpha-GlcNAc binds to hen lysozyme in both alpha- and beta-modes, which correspond to the binding orientation of alpha-GlcNAc and that of beta-GlcNAc, respectively, as determined by X-ray crystallographic studies, but it binds predominantly in the beta-mode below about pH 4, (2) beta-GlcNAc binds to hen and turkey lysozymes predominantly in the beta-mode above about pH 4 and in both alpha- and beta-modes below pH 4, and (3) alpha-GlcNAc binds to turkey lysozyme predominantly in the beta-mode over the whole pH range studied.     

Human liver aldehyde reductase: pH dependence of steady-state kinetic parameters.

The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V/K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2'-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2'-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V/K for NADPH, the V/K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2'-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V/K for DL-glyceraldehyde and V/K for L-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as D-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V/K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V/K for 3-APADPH, and V/K for D-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.