Inhibition of wild-type and mutant neuronal nicotinic acetylcholine receptors by local anesthetics.

Inhibition of neuronal nicotinic receptors can be regulated by the presence of specific amino acids in the beta subunit second transmembrane domain (TM2) domain. We show that the incorporation of a mutant beta4 subunit, which contains sequence from the muscle beta subunit at the TM2 6' and 10' positions of the neuronal beta4 subunit, greatly reduces the sensitivity of receptors to the local anesthetic [2-(triethylamino)-N-(2,6-dimethylphenyl)acetamide] (QX-314). Although differing in potency, the inhibition of both wild-type alpha3beta4 receptors and alpha3beta4(6'F10'T) receptors by QX-314 is voltage-dependent and noncompetitive. Interestingly, the potency of the local anesthetic tetracaine for the inhibition of alpha3beta4 and alpha3beta4(6'F10'T) receptors seems unchanged when measured at -50 mV. However, whereas the onset of inhibition of wild-type alpha3beta4 receptors is voltage-dependent and noncompetitive, the onset of inhibition of alpha3beta4(6'F10'T) receptors by tetracaine is unaffected by membrane voltage, and at concentrations < or = 30 microM seems to be competitive with acetylcholine. This may be due to either direct effects of tetracaine at the acetylcholine binding site or preferential block of closed rather than open channels in the mutant receptors. Further analysis of receptors containing the 6' mutation alone suggests that although the 6' mutation is adequate to alter the voltage dependence of tetracaine inhibition, both point mutations are required to produce the apparent competitive effects.

Primary 13C and beta-secondary 2H KIEs for trans-sialidase. A snapshot of nucleophilic participation during catalysis.

Trypanosoma cruzi trans-sialidase catalyzes a novel reaction that involves the transfer of sialic acid between host and parasite glycoconjugates. In this paper, we report kinetic isotope effect studies on recombinant trans-sialidase. beta-Dideuterium and primary 13C isotope effects were measured for a good substrate, sialyl-lactose, and a slow substrate, sialyl-galactose, in both acid-catalyzed solvolysis and enzymatic transfer reactions. The beta-dideuterium isotope effect for sialyl-lactose in the acid hydrolysis reaction was 1.113 +/- 0.012. The primary 13C isotope effects for hydrolysis of sialyl-lactose and sialyl-galactose were 1. 016 +/- 0.011 and 1.015 +/- 0.008, respectively. In the enzymatic transfer reactions, the beta-dideuterium and primary 13C effects for sialyl-galactose were 1.060 +/- 0.008 and 1.032 +/- 0.008, respectively. The isotope effects for hydrolysis describe a dissociative SN1-like mechanism, and these data are contrasted by the data for the enzyme-catalyzed reaction. The enzymatic deuterium isotope effects are lower by a factor of 2, but the primary carbon isotope effects are higher by a factor of 2. This pattern describes a mechanism involving nucleophilic participation in the rate-determining transition state.

Use of an altered sugar-nucleotide to unmask the transition state for alpha(2-->6) sialyltransferase.

Rat liver alpha(2-->6) sialyltransferase catalyzes the formation of a glycosidic bond between N-acetylneuraminic acid and the 6-hydroxyl group of a galactose residue at the nonreducing terminus of an oligosaccharide. This reaction has been investigated through the use of the novel sugar-nucleotide donor substrate UMP-NeuAc. A series of UMP-NeuAc radioisotopomers were prepared by chemical deamination of the corresponding CMP-NeuAc precursors. Kinetic isotope effects (KIEs) on V/K were measured using mixtures of radiolabeled UMP-NeuAc's as the donor substrate and N-acetyllactosamine as the acceptor. The secondary beta-(2)H KIE was 1.218 +/- 0.010, and the primary (14)C KIE was 1.030 +/- 0.010. A large inverse (3)H binding isotope effect of 0.944 +/- 0.010 was measured at the terminal carbon of the NeuAc glycerol side chain. These KIEs observed using UMP-NeuAc are much larger than those previously measured with CMP-NeuAc [Bruner, M., and Horenstein, B. A. (1998) Biochemistry 37, 289-297]. Solvent deuterium isotope effects of 1.3 and 2.6 on V/K and V(max) were observed with CMP-NeuAc as the donor, and it is revealing that these isotope effects vanished with use of the slow donor substrate UMP-NeuAc. Bell-shaped pH versus rate profiles were observed for V(max) (pK(a) values = 5.5, 9.0) and V/K(UMP)(-)(NeuAc) (pK(a)values = 6.2, 9.0). The results are considered in terms of a mechanism involving an isotopically sensitive conformational change which is independent of the glycosyl transfer step. The isotope effects reveal that the enzyme-bound transition state bears considerable charge on the N-acetylneuraminic acid residue, and this and other features of this mechanism provide new directions for sialyltransferase inhibitor design.

Sensitivity to voltage-independent inhibition determined by pore-lining region of the acetylcholine receptor.

Some noncompetitive inhibitors (e.g., ganglionic blockers) exhibit selectivity for the inhibition of neuronal nicotinic acetylcholine receptors (nAChRs). This study characterizes the mechanism of selective long-term inhibition of neuronal and muscle-neuronal chimeric nAChRs by bis(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (bis-TMP-10 or BTMPS), a bifunctional form of the potent ganglionic blocker tetramethylpiperidine. Long-term inhibition of neuronal nAChRs by bis-TMP-10 has been previously demonstrated to arise, at least in part, from the binding of the bis compound to neuronal beta-subunits. In this study, long-term inhibition is demonstrated to be dependent upon the presence of sequence element(s) within the pore-lining second transmembrane domain (tm2) of neuronal beta-subunits; however, the inhibitor binding site itself does not appear to be contained within the segment of the channel pore influenced by the membrane electric field. Specifically, our results imply that bis-TMP-10 interacts with an activation-sensitive element, the availability of which may be regulated by a sequence in the tm2 domain. Furthermore, we demonstrate a compound length requirement for long-term inhibition that would be consistent with binding to multiple sites located on the extracellular portion of the receptor.

Isotope trapping and kinetic isotope effect studies of rat liver alpha-(2-->6)-sialyltransferase.

A mechanistic study of rat liver alpha-(2-->6) sialyltransferase (ST) is presented that includes isotope trapping experiments and kinetic isotope effects on V/K for the ST-catalyzed reaction of isotopically labeled CMP-N-acetylneuraminate and N-acetyllactosamine. The isotope trapping experiments confirmed that the kinetic mechanism is steady-state random, and further analysis indicated that for this sialyltransferase the experimentally observed isotope trapping ratio (product trapped/substrate released) was equivalent to the commitment to catalysis, Cf, the quantity required to correct the kinetic isotope effects. Cf was found to range from 1.0 (at 1.6 mM LacNAc) to 1.7 (at 100 mM LacNAc). After correction for Cf, the isotope effects were as follows: secondary beta-dideuterium, 1.04-1. 05; anomeric carbon primary 14C, 1.000 +/- 0.004; a small 3H binding effect of 1.016 +/- 0.007 at C9; and a carboxylate carbon secondary 14C isotope effect of 0.998 +/- 0.004. This pattern of KIEs is quite different than observed for solvolysis of CMP-NeuAc [Horenstein, B. A., and Bruner, M. (1996) J. Am. Chem. Soc. 118, 10371-10379]. Based on the results of ab-initio modeling of isotope effects, a hypothesis is presented which reconciles the unusual pattern of KIEs on the basis of binding interactions at the carboxylate carbon.

Amidrazone analogues of D-ribofuranose as transition-state inhibitors of nucleoside hydrolase.

The transition state of inosine during hydrolysis by nucleoside hydrolase has been characterized by kinetic isotope effects, bond-energy/bond-order vibrational analysis, and molecular electrostatic potential surface calculations [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795; Horenstein, B. A., & Schramm, V. L. (1993) Biochemistry 32, 7089-7097]. The heterocyclic base is protonated and the anomeric carbon of the ribofuranosyl ring is flattened to form a transition-state with extensive oxocarbenium ion character. With their delocalized charge and flattened structures, amidrazone analogues of D-ribofuranose provide both geometric and electronic mimics of the ribosyl group at the transition-state of nucleoside hydrolase. A family of riboamidrazones was synthesized with H, phenyl, and p-nitrophenyl N-substituents. The analogues were competitive inhibitors with respect to inosine and gave Ki values of 10(-5), 2 x 10(-7), and 1 x 10(-8) M, respectively. (p-Nitrophenyl)riboamidrazone exhibited slow-onset, tight-binding inhibition, with an overall dissociation constant of 2 x 10(-9) M. The binding is reversible with an off-rate of 3 x 10(-3) s-1. Tight binding can be attributed to the close spatial match between the molecular geometry of (p-nitrophenyl)riboamidrazone and the transition-state stabilized by nucleoside hydrolase. The favorable binding interactions of the (p-nitrophenyl)riboamidrazone include oxocarbenium ion mimicry, isosteric ribosyl hydroxyls, and hydrophobic and H-bonding interactions at the nitrophenyl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of the molecular electrostatic potential surface of an enzymatic transition state with novel transition-state inhibitors.

The transition state stabilized by nucleoside hydrolase from Crithidia fasciculata is characterized by nearly complete glycosidic bond cleavage and oxycarbonium character in the ribosyl group [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. The electrostatic potential surface of the transition state provides detailed information which should be useful in the design of transition-state analogues [Horestein, B. A., & Schramm, V. L. (1993) Biochemistry 32, 7089-7097]. The electrostatic potential surface of inosine at the transition state contains a distributed positive charge resulting from the oxycarbonium ion character of the ribosyl ring. The ribosyl ring pucker is 3'-exo as a result of the near sp2 hybridization at Cl' of the ribose ring. A series of transition-state analogues have been synthesized which incorporate single or combined features of the transition state. Each feature of the transition state was analyzed for its contribution to binding energy. Kinetic inhibition constants correlate with the similarity of the inhibitor to the experimentally determined transition-state structure. Dissociation constants for the substrate and products of the reaction of inosine, hypoxanthine, and ribose are 380, 6200, and 700 microM, respectively. A transition-state analogue was synthesized which contains the required hydroxyl groups of the ribose ring, the positive charge feature of the oxycarbonium ion, and a hydrophobic mimic of the purine ring. The inhibitor 1(S)-phenyl-1,4-dideoxy-1,4-iminoribitol acts as a competitive inhibitor with respect to inosine with a dissociation constant of 0.17 microM. In addition, the inhibitor exhibits slow-onset inhibition which provides a final equilibrium dissociation constant of approximately 0.03 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Electronic nature of the transition state for nucleoside hydrolase. A blueprint for inhibitor design.

A new approach to understanding transition-state structure is presented which involves the sequential application of experimental and computational methods. A family of experimentally determined kinetic isotope effects is fit simultaneously in a vibrational analysis to provide a geometric model of the transition state. The electrostatic potential surface of the geometric model is defined by molecular orbital calculations to detail the electronic nature of the transition state. The method provides both geometric and charge information for the enzyme-stabilized transition state. Electrostatic potential surface calculations were applied to the N-glycohydrolase reaction catalyzed by nucleoside hydrolase from the trypanosome Crithidia fasciculata. A geometric model of the transition-state structure for the enzymatic hydrolysis of inosine by nucleoside hydrolase has been established by the analysis of a family of kinetic isotope effects [Horenstein, B.A., Parkin, D.W., Estupinan, B., & Schramm, V.L. (1991) Biochemistry 30, 10788]. The transition state has substantial oxycarbonium ion character, but the results of electrostatic potential calculations indicate that the transition-state charge is distributed over the ribosyl ring rather than existing as a localized C+-O<==>C = O+ resonance pair. The electrostatic potential surfaces of the substrate and enzyme-bound products differ considerably from that of the transition state. At the transition state both hypoxanthine and ribose demonstrate regions of positive charge. The positive charge on the ribosyl oxycarbonium ion is moderated by association with an enzyme-directed water nucleophile. The enzyme-bound products contain adjacent areas of negative charge. The electrostatic potential surfaces provide novel insights into transition-state structure and the forces causing release of products.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism.

Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.

Transition-state analysis of nucleoside hydrolase from Crithidia fasciculata.

The transition state of nucleoside hydrolase from the trypanosome Crithidia fasciculata has been characterized by multiple Vmax/Km kinetic isotope effects with labeled inosine and adenosine as substrates. Nucleoside hydrolase catalyzes the hydrolysis of the N-glycosidic linkage of the commonly occurring purine and pyrimidine nucleosides, with Vmax/Km ranging over 2 orders of magnitude. The kinetic isotope effects for inosine were [1'-3H] = 1.150 +/- 0.006, [2'-3H] = 1.161 +/- 0.003, [1'-14C] = 1.044 +/- 0.004, [9-15N] = 1.026 +/- 0.004, [4'-3H] = 0.992 +/- 0.003, and [5'-3H] = 1.051 +/- 0.003. The magnitude of the kinetic isotope effects for inosine, an equivalent [1'-3H] kinetic isotope effect for the poor substrate adenosine, and the rapid equilibrium random kinetic mechanism [Parkin D, W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., & Schramm, V. L. (1991) J. Biol. Chem. (in press)] all indicate that the isotope effects are fully expressed. The kinetic and solvent deuterium isotope effects have been used to analyze the transition-state structure using bond energy bond order vibrational analysis. The transition state involves a protonated hypoxanthine leaving group with a C-N glycosidic bond elongated to approximately 2 A. The ribose group contains substantial carbocationic character, unusually strong hyperconjugation of H2', and a bond length of approximately 3 A to the incoming oxygen nucleophile. The remote isotope effect (4'-3H and 5'-3H) and the results of transition-state calculations provide the most detailed description of the steric and bonding properties of an enzyme-stabilized transition state.

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects.

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)