Chiral nature of covalent methylphosphonyl conjugates of acetylcholinesterase

This paper examines the chiral nature of the covalent conjugates formed upon reaction of acetylcholinesterase (AchE) with enantiomeric cycloheptyl, isopropyl, and 3,3-dimethylbutyl methylphosphonyl thiocholines. With the exception of the conjugate formed from reaction of AchE with RP-cycloheptyl methylphosphonyl thiocholine, all enantiomeric conjugates underwent oxime reactivation at rates that were within 2-3-fold of each other. Oxime reactivation was, therefore, independent of both initial configuration about phosphorus and the alkyl phosphonyl ester (-OR) moiety. Aging of the enantiomeric cyclopheptyl and isopropyl methylphosphonyl conjugates occurred exclusively for the conjugate formed from the SP-enantiomer and therefore displayed an absolute dependence on the initial configuration of the methylphosphonyl group. Equilibrium titrations with decidium, a fluorescent bisquaternary competitive inhibitor of AchE, provided an index of aging and enantiomeric configuration of the conjugates independent of enzyme activity. Decidium association with the enantiomeric conjugates (prior to aging) showed no marked dependence on the initial configuration about phosphorus but was measurably dependent on nature of the -OR moiety. These results are interpreted with respect to symmetry and nonrigidity of the organophosphonyl conjugates and are consistent with formation of final methylphosphonyl conjugates that are enantiomerically pure and of opposite configuration. These studies indicate that the active center of AchE comprises at least two kinetically distinct environments separate from the esteratic region but located within 5 A of the nucleophilic serine and differing in dipolar characteristics that promote charge separation and general acid catalysis.     

Chiral reactions of acetylcholinesterase probed with enantiomeric methylphosphonothioates. Noncovalent determinants of enzyme chirality

Enantiomeric cycloheptyl- and isopropyl methylphosphonothioates containing uncharged and cationic leaving groups, and 3,3-dimethylbutyl methylphosphonyl thiocholines were synthesized, and their inhibition of acetylcholinesterase from Torpedo examined. Bimolecular inhibition constants spanned 10(1)-10(9) M-1.min-1, equilibrium dissociation constants 10(-3)-10(-7) M, and phosphonylation constants 1-300 min-1. A general but not absolute preference for the SP-enantiomer, in the range 170-4600 for cycloheptyl-, 0.6-150 for isopropyl-, and 30 for 3,3-dimethylbutyl methylphosphonothioates, varied with nature of the alkyl ester (-OR) and thioic leaving groups (-SR') surrounding phosphorus. While the overall bimolecular reaction constant showed no marked dependence on ionic strength of the medium, the microscopic kp and KD for the RP- but not SP-cycloheptyl methylphosphonyl thiocholine underwent marked reduction with decreases in ionic strength. This result unmasks the interplay between occupation of the active center and productivity of that occupation. These studies reveal that chiral reactions with acetylcholinesterase are dependent more on the nature of the groups surrounding the tetrahedral phosphorus than on the absolute configuration about the phosphorus atom and indicate that the active center comprises partially overlapping subsites that can accommodate the -OR and -SR' groups. The presence of neighboring subsites characterized by different steric, electrostatic, and hydrophobic properties permits a multiplicity of binding orientations, independent of chiral configuration, and which account for the large variation in chiral preference seen among organophosphonates containing different substituents.     

Effect of homologous series of n-alkyl sulfates and n-alkyl trimethylammonium bromides on low molecular mass protein tyrosine phosphatase activity

The effect of anionic and cationic surfactants on acid phosphatase denaturation has been extensively studied. Low molecular mass (LMr) protein tyrosine phosphatase (PTP), a key regulatory enzyme involved in many different processes in the cell, was distinctly affected by anionic (homologous series of n-alkyl sulfates (C8-C14)) and cationic (n-alkyl trimethylammonium bromides (C12-C16)) surfactants. At concentrations 10-fold lower critical micellar concentration (cmc) values, the enzyme was completely inactivated in the presence of anionic surfactants, in a process independent of the pH, and dependent on the chain length of the surfactants. Under the same conditions, the effect of cationic surfactants on the enzyme activity was pH-dependent and only at pH 7.0 full inactivation was observed at concentrations 10-fold higher cmc values. In contrast to cationic surfactants the effect of anionic surfactants on the enzyme activity was irreversible and was not affected by the presence of NaCl. Inorganic phosphate, a known competitive inhibitor of PTP, protected the enzyme against inactivation by the surfactants. Our results suggest that the inactivation of the LMr PTP by anionic and cationic surfactants involved both electrostatic and hydrophobic interactions, and that the interactions enzyme-surfactants probably occurred at or near the active site.     

Mechanism of oxime reactivation of acetylcholinesterase analyzed by chirality and mutagenesis

Organophosphates inactivate acetylcholinesterase by reacting covalently with the active center serine. We have examined the reactivation of a series of resolved enantiomeric methylphosphonate conjugates of acetylcholinesterase by two oximes, 2-pralidoxime (2-PAM) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium) (HI-6). The S(p) enantiomers of the methylphosphonate esters are far more reactive in forming the conjugate with the enzyme, and we find that rates of oxime reactivation also show an S(p) versus R(p) preference, suggesting that a similar orientation of the phosphonyl oxygen toward the oxyanion hole is required for both efficient inactivation and reactivation. A comparison of reactivation rates of (S(p))- and (R(p))-cycloheptyl, 3,3-dimethylbutyl, and isopropyl methylphosphonyl conjugates shows that steric hindrance by the alkoxy group precludes facile access of the oxime to the tetrahedral phosphorus. To facilitate access, we substituted smaller side chains in the acyl pocket of the active center and find that the Phe295Leu substitution enhances the HI-6-elicited reactivation rates of the S(p) conjugates up to 14-fold, whereas the Phe297Ile substitution preferentially enhances 2-PAM reactivation by as much as 125-fold. The fractional enhancement of reactivation achieved by these mutations of the acyl pocket is greatest for the conjugated phosphonates of the largest steric bulk. By contrast, little enhancement of the reactivation rate is seen with these mutants for the R(p) conjugates, where limitations on oxime access to the phosphonate and suboptimal positioning of the phosphonyl oxygen in the oxyanion hole may both slow reactivation. These findings suggest that impaction of the conjugated organophosphate within the constraints of the active center gorge is a major factor in influencing oxime access and reactivation rates. Moreover, the individual oximes differ in attacking orientation, leading to the presumed pentavalent transition state. Hence, their efficacies as reactivating agents depend on the steric bulk of the intervening groups surrounding the tetrahedral phosphorus.     

Differential reactivity of the two active site cysteine residues generated on reduction of pig heart lipoamide dehydrogenase.

Reduction of the active center disulfide bond in the flavoprotein pig heart lipoamide dehydrogenase generates two sulfur moieties which are chemically inequivalent in the 2-electron reduced form of the enzyme. Thus 1 cysteine residue is at least 13-fold more reactive than its partner toward iodoacetamide at pH 7.6. This selectivity was demonstrated by reaction of the 2-electron reduced enzyme with a low concentration of iodo[1-14C]acetamide under anaerobic conditions. The formation of a monolabeled derivative is accompanied by the reappearance of a spectrum of oxidized bound flavin, clearly different from that of the native enzyme. Alkylation of the remaining cysteine residues with iodo[12C]acetamide enabled the isolation of a tryptic version of the active center disulfide peptide. A single chymotryptic cleavage between the 2 alkylated cysteine residues generated a cationic and an anionic fragment containing 7% and 93% of the radioactivity of the purified tryptic peptide, respectively. The monolabeled derivative is catalytically inactive toward reduced or oxidized lipoamide, but is approximately 2-fold better as a transhydrogenase than the native protein using NADH and acetylpyridine adenine dinucleotide as substrates. Anaerobic titration with NADH leads to reduction of the flavin with concomitant formation of long wavelength absorption of low intensity. No intermediate reduced states were detected in this titration analogous to the red 2-electron form observed with the native enzyme. Similarly, intermediates during reduction of the enzyme by 1 eq of dithionite have not been detected.     

Mutant cholinesterases possessing enhanced capacity for reactivation of their phosphonylated conjugates

Selective mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7) phosphonylated with chiral S(P)- and R(P)-cycloheptyl, -3,3-dimethylbutyl, and -isopropyl methylphosphonyl thiocholines were subjected to reactivation by the oximes HI-6 and 2-PAM and their reactivation kinetics compared with wild-type AChE and butyrylcholinesterase (EC 3.1.1.8). Mutations in the choline binding site (Y337A, Y337A/F338A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) were employed to enlarge active center gorge dimensions. HI-6 was more efficient than 2-PAM (up to 29000 times) as a reactivator of S(P)-phosphonates (k(r) ranged from 50 to 13000 min(-1) M(-1)), while R(P) conjugates were reactivated by both oximes at similar, but far slower, rates (k(r) < 10 min(-1) M(-1)). The Y337A substitution accelerated all reactivation rates over the wild-type AChE and enabled reactivation even of R(P)-cycloheptyl and R(P)-3,3-dimethylbutyl conjugates that when formed in wild-type AChE are resistant to reactivation. When combined with the F295L or F297I mutations in the acyl pocket, the Y337A mutation showed substantial enhancements of reactivation rates of the S(P) conjugates. The greatest enhancement of 120-fold was achieved with HI-6 for the F295L/Y337A phosphonylated with the most bulky alkoxy moiety, S(P)-cycloheptyl methylphosphonate. This significant enhancement is likely a direct consequence of simultaneously increasing the dimensions of both the choline binding site and the acyl pocket. The increase in dimensions allows for optimizing the angle of oxime attack in the spatially impacted gorge as suggested from molecular modeling. Rates of reactivation reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in protection and treatment of organophosphate exposure.     

General occurrence of binding to acetylcholinesterase-substrate complex in noncompetitive inhibition and in inhibition by substrate

To assess the relative importance of binding to enzyme-substrate complex (E.S) and to acetylenzyme (EA), noncompetitive inhibition has been studied in hydrolysis by acetylcholinesterase (AcChE) of cationic and uncharged substrates - acetylcholine (AcCh), 3,3-dimethylbutyl acetate, n-butyl acetate, 2-(methylammonio)ethyl acetate, 2- (N,N-diethyl-N-n-butylammonio)ethyl acetate (DEBAAc) and 2-(methylsulfonyl)ethyl acetate. For the N-trimethyl quaternary ions related to AcCh, tetramethylammonium ion, choline and choline ethyl ether, noncompetitive inhibition (Ki(nonc) is more favorable with the slower substrates than with AcCh, i.e., when E.S greater than EA, and is attributed to formation of enzyme-substrate-inhibitor complexes, E.S.I'. Noncompetitive inhibition by tetraethyl-, tert-butyl- and isopropylammonium ions, and acetamidocholine and its lower dimethyl analogue, is also attributed to E.S.I' complexes. Peripheral binding of these inhibitors decreases acylation more than deacylation. Some tertiary dimethylamonio ions have more favorable Ki(nonc) values with AcCh, decreasing deacylation more than acylation. The substrate DEBAAc is a more effective noncompetitive than competitive inhibitor in hydrolysis of AcCh, indicating that it binds more strongly in a peripheral site than in the active site of the free enzyme. In its hydrolysis by AcChE, it acts as its own noncompetitive inhibitor, by this non-productive binding. Formation of E.S.I' complexes is a general characteristic of hydrolysis by AcChE and decrease in rates at high concentrations of AcCh and related substrates is attributed to peripheral regulatory site binding, formation of E.S.S' complexes, rather than to binding to the acetylenzyme.     

Chemical modification of lysine residues of beta-1,3-glucanase from Spisula sachalinensis

The effects of chemical modification of the amino groups of lysine residues on the activity of beta-1.3-glucanase from Spisula sachalinensis were studied. Modification of two lysine residues per molecule did not affect either the enzyme activity with respect to laminarine, nor the Km value. The modified beta-1.3-glucanase retains the ability to catalyze the transglycosylation and cleaves the high molecular weight CM-pachyman at the same rate as does the native enzyme. No significant changes in the enzyme thermal stability were observed. Thus, the modified enzyme groups cannot be involved in the enzyme active center and are exposed on the surface of the protein globule. The chemical modification was shown to have no effect on the enzyme kinetics, which is essential for its immobilization.     

Affinity chromatographic sorting of carboxypeptidase a and its chemically modified derivatives

Carboxypeptidase A and derivatives obtained by chemical modification of various active center components were subjected to affinity chromatography on a p-aminobenzylsuccinic acid-Sepharose 4B conjugate. Tetardation of the enzyme on the column was dependent on the residue modified when elution was carried out with 0.3 m NaCl at pH 7.0. Both the functional zinc atom and the active site residue Glu-270 are essential for effective adsorption while alteration of residues involved in hydrophobic interaction with substrate or in recognition of its terminal carboxyl group decreased retention on the affinity matrix. Elution of native carboxypeptidase with competing soluble benzylsuccinic acid indicated that only active center binding of the immobilized inhibitor accounts for retardation of the enzyme on the column. Hence, affinity chromatography on this biospecific adsorbent using mild elution conditions (which do not distort protein structure) provides an excellent tool for the rapid isolation and purification of active center modified enzyme even from a complex mixture of reaction products.     

Thrombolytic action of urokinase preparation covalently bound to modified thrombin

Urokinase was covalently bounded with modified thrombin. Thrombin was modified by carbodiimide and 1, 12-dodecamethylenediamine. In this conjugate thrombin is not catalytically active and does not induce platelets aggregation. The catalytic properties of modified urokinase do not essentially differ from native enzyme but its thermostability increases. The modified urokinase thrombolytic effect is at least 10-fold higher than the native one. In femoral arteries of experimental thrombosis the conjugate urokinase-thrombin brings about total thrombolytic effect as early as 1.5 hours after injection (2500 IU per 1 kg of the animals weight). The causes of the observed effect were discussed.     

beta-Alanine synthase: purification and allosteric properties.

beta-Alanine synthase has been purified greater than 1000-fold to homogeneity from rat liver. The enzyme has a subunit molecular weight of 42,000 and a native size of hexamer. The enzyme undergoes ligand-induced changes in polymerization: association in response to the substrate, N-carbamoyl-beta-alanine, and the inhibitor, propionate; and dissociation in response to the product, beta-alanine. The ability of the substrate to associate the pure native enzyme to a larger polymeric species was exploited in the final purification step. The purified enzyme had a pI of 6.7, a Km of 8 microM, and a kcat/Km of 7.9 x 10(4) M-1 s-1. Positive cooperativity was observed toward the substrate N-carbamoyl-beta-alanine, with nH = 1.9. Such cooperativity occurred at substrate concentrations below 12 nM, so that this activation most likely occurs at a regulatory site, with a significantly stronger affinity for N-carbamoyl-beta-alanine than that shown by the catalytic site. The enzyme was sensitive to denaturation, which could be minimized by avoiding heat steps during the purification and by the presence of reducing agents. Such denatured enzyme had little change in Vmax, but had much higher Km, and had also lost the ability to associate or dissociate in response to effectors. After purification, enzyme stability was achieved by the addition of glycerol and detergent.     

Ligand exclusion on acetylcholinesterase

This paper examines covalent reactivity of AchE with respect to cationic and uncharged methylphosphonates and substrates in the absence and presence of cationic ligands selective for the active center and the peripheral anionic site. The organophosphorus inhibitors are enantiomeric alkyl methylphosphonothioates (1-5) containing cycloheptyl and isopropyl phosphono ester groups and S-methyl, S-n-pentyl, and S-[beta-(trimethylammonio)ethyl] leaving groups; these agents differ in their configuration about phosphorus and their steric, hydrophobic, and electrostatic characteristics. The synthetic substrates examined are acetylthiocholine, p-nitrophenyl acetate, and 7-acetoxy-4-methylcoumarin (7AMC). Antagonism of the methylphosphonothioate reaction by cationic ligands is strongly dependent on the nature of both the cation and the methylphosphonate but independent of the configuration about phosphorus. While all cations cause linear mixed inhibition of acetylthiocholine hydrolysis, there are observed a variety of inhibition patterns of 7AMC and p-nitrophenyl acetate hydrolysis that are distinctly nonlinear, as well as patterns in which the reciprocal plots intersect in the upper right quadrant. Strong antagonism of cationic (methylphosphonyl)thiocholines correlates very well with linear inhibition of acetylthiocholine. Ligands that cause only negligible antagonism of the uncharged methylphosphonates display nonlinear inhibition of uncharged substrates. These relationships, since they are most pronounced for peripheral site ligands and are strongly dependent on the charge carried by the reactant, suggest that the peripheral anionic site alters enzyme reactivity through an electrostatic interaction with the net negative active center. Such behavior indicates a potential role for the peripheral anionic site in conserving AchE catalytic efficiency within a narrow range of values.     

Dimensional mapping of the active site of cholesterol esterase with alkylboronic acid inhibitors

The cholesterol esterase-catalyzed hydrolysis of the water-soluble substrate p-nitrophenyl butyrate occurs via an acylenzyme mechanism, and is competitively inhibited by boronic acid transition state analog inhibitors. Accordingly, we undertook to dimensionally map the enzyme's active site via synthesis and characterization of a series of n-alkyl boronic acid inhibitors. The most potent of these is n-hexaneboronic acid, with a Ki = 13 +/- 1 microM, since inhibitor potency declines for both longer and shorter boronic acids. No inhibition is observed for methaneboronic acid and n-octaneboronic acid inhibits poorly, with a Ki of 7 mM. These results indicate that the ability of the enzyme to form tight complexes with boron-containing transition state analog inhibitors is sensitive to alkyl chain length. The trend in inhibitor potency is discussed in terms of substrate specificity of and transition state stabilization by cholesterol esterase, and has important implications for the design of optimal reversible inhibitors of the enzyme.     

Fractional diffusion-limited component of reactions catalyzed by acetylcholinesterase

The values of kcat/Km for the reactions of four substrates, p-nitrophenyl acetate (PNPA), propionyl-beta-methylthiocholine (PrMSCh), 3,3-dimethylbutyl thioacetate (DBTA), and acetylthiocholine (AcSCh), with acetylcholinesterase were determined as a function of increasing viscosity (eta rel) in sucrose-containing and in glycerol-containing buffers. Glycerol, or possibly some contaminant of it, was found to be a nonspecific inhibitor and sucrose a nonspecific activator of the enzyme as reflected in the dependence of kcat/Km values measured for PNPA and PrMSCh upon the concentration of these reagents. The rates of reactions of these two substrates, the first neutral and the second cationic, are chemically limited rather than diffusion limited, and they thus serve as quantitative controls or internal standards to monitor the effects of the viscosogens on the enzyme, which are not related to diffusion. The additional effect on kcat/Km over the controls observed for the rapidly reacting substrates AcSCh (cationic) and DBTA (neutral) serves as a measure of the extent to which these values of kcat/Km measure diffusion-controlled processes. The reaction rate of DBTA with the enzyme is 24% diffusion controlled as measured in glycerol-containing buffers and 16-20% as determined in sucrose-containing buffers, while that for AcSCh is 100% (in glycerol) and 24-40% (in sucrose) diffusion controlled.     

Endo-beta-1,3-glucanase using affinity inhibition

Synthetic 2',3'-epoxypropyl-1-thio-beta-D-glucopyranoside selectively modifies a catalytically essential nucleophylic group in the active site of beta-1,3-glucanase LIV from the marine mollusc Spisula sachalinensis, the inactivation being as high as 95%. The properties of native and epoxypropylthioglucopyranoside-inhibited glucanase LIV were compared, using UV-spectroscopy, SDS polyacrylamide gel electrophoresis and isoelectrofocusing. It was found that the addition of laminarine and laminarioligosaccharides to a solution of the inhibited enzyme induces UV-differential spectra typical for the tryptophanyl residue involved in the formation of the enzyme-inhibitor-substrate complex. The glucone-1,5-lactone does not produce such spectrum. It was shown that epoxypropylthioglucopyranoside protects the accessible tryptophanyl residues in the enzyme active center against the oxidation by N-bromosuccinimide.     

Anchimeric assistance in the intramolecular reaction of glucose-dehydrogenase-polyethylene glycol NAD conjugate

Polyethylene glycol-bound derivatives of NAD(P) (PEG-NAD(P)) are water-soluble macromolecular coenzymes used in continuous enzyme reactors. These NAD(P) derivatives have good coenzyme activity for many dehydrogenases, but some enzymes such as glucose dehydrogenase (EC 1.1.1.47) show very low activity with these derivatives (less than 0.1% of that for native NAD(P)). In this work, we prepared a covalently linked glucose-dehydrogenase-polyethylene glycol-NAD conjugate (GlcDH-PEG-NAD) and found that the conjugate shows a much higher reaction rate than that of the native enzyme plus PEG-NAD: the ratio of the reaction rates of GlcDH-PEG-NAD and the native enzyme plus PEG-NAD is calculated to be 10,000-fold at the concentrations of the enzyme subunit and NAD moiety of 0.31 and 0.65 microM, respectively; the rate of the conjugate is even higher than that of the native enzyme plus native NAD. This rate acceleration is due to the increase in the effective concentration of NAD moiety ("anchimeric assistance") and demonstrates the potential of covalent linking for improving the interaction between an enzyme and a coenzyme derivative.     

Catalytic and immunochemical properties of ferritin conjugates with horseradish peroxidase

The human spleen ferritin--horseradish peroxidase conjugate (HRP--Fer) was synthesized by periodate oxidation of the enzyme carbohydrate fragment. The protein fraction containing 1-2 peroxidase molecules and characterized by kinetic homogeneity was obtained in the peroxidatic ortho-dianisidine (o-DA) oxidation reaction. Gel diffusion precipitation of HRP--Fer with peroxidases and ferritin antibodies was carried out. The precipitation confirms the retention by peroxidase and ferritin of their antigenic properties. The kinetics of peroxidatic oxidation of o-DA by the HRP--Fer conjugate was studied within the temperature interval of 15-37 degrees C. The value of catalytic constant for this reaction exceeds that for native peroxidase 1.75-fold. A kinetic analysis of thermal inactivation of peroxidase and its conjugate was performed within the temperature range of 40-65 degrees C. The effective rate constants of inactivation obtained from the first order equation are higher for HRP--Fer than for the native enzyme. The effect of pH on the rates of inactivation of HRP--Fer and the non-modified enzyme was studied at 50 degrees C. The enzyme and its conjugate were shown to stabilize in acid media. The HRP--Fer conjugate can be used as an effective tool in immunoenzymatic assays of ferritin.     

Purification and properties of a phosphohydrolase from Enterobacter aerogenes.

A phosphohydrolase from Enterobacter aerogenes which hydrolyzes phosphate mono- and diesters has been purified approximately 50-fold to apparent homoeneity and crystallized. The enzyme is produced when the bacteria utilize phosphate diesters as sole phosphorus source. From sedimentation equilibrium experiments the molecular weight of the native enzyme is 173,000; from sodium dodecyl sulfate polyacrylamide gel electrophoresis the subunit molecular weight is 29,000, indicating that the enzyme is hexameric. The hydrolytic activity of the enzyme using both mono- and diesters is maximal at pH 5; THE Km of the enzyme for bis-p-nitrophenyl phosphate is constant from pH 5 to 8.5 whereas that for p-nitrophenyl phosphate increases about 40-fold as the pH increases over the same range. The phosphodiesterase activity is not inhibited by chelating agents but is inhibited by several divalent metal ions. 31-P NMR spectroscopy was used to identify the hydrolysis products of glycoside cyclic phosphates. The enzyme-catalyzed hydrolysis of methyl beta-D-ribofuranoside cyclic 3:5-phosphate yields exclusively the 5-phosphate whereas that of adenosine 3:5-monophosphate yields a 4:1 mixture of 3- and 5- AMP.     

Interaction kinetics of reversible inhibitors and substrates with acetylcholinesterase and its fasciculin 2 complex

Fasciculin 2 (Fas2), a three-fingered peptide of 61 amino acids, binds tightly to the peripheral site of acetylcholinesterases (AChE; EC ), occluding the entry portal into the active center gorge of the enzyme and inhibiting its catalytic activity. We investigated the mechanism of Fas2 inhibition by studying hydrolysis of cationic and neutral substrates and by determining the kinetics of interaction for fast equilibrating cationic and neutral reversible inhibitors with the AChE.Fas2 complex and free AChE. Catalytic parameters, derived by eliminating residual Fas2-resistant activity, reveal that Fas2 reduces k(cat)/K(m) up to 10(6)-fold for cationic substrates and less than 10(3)-fold for neutral substrates. Rate constants for association of reversible inhibitors with the active center of the AChE.Fas2 complex were reduced about 10(4)-fold for both cationic and neutral inhibitors, while dissociation rate constants were reduced 10(2)-to 10(3)-fold, compared with AChE alone. Rates of ligand association with both AChE and AChE.Fas2 complex were dependent on the protonation state of ionizable ligands but were also markedly reduced by protonation of enzyme residue(s) with pK(a) of 6.1-6.2. Linear free energy relationships between the equilibrium constant and the kinetic constants show that Fas2, presumably through an allosteric influence, markedly alters the position of the transition state in the reaction pathway. Since Fas2 complexation introduces an energetic barrier for hydrolysis of substrates that exceeds that found for association of reversible ligands, Fas2 influences catalytic parameters by a more complex mechanism than simple restriction of diffusional entry and exit from the active center. Conformational flexibility appears critical for facilitating ligand passage in the narrow active center gorge for both AChE and the AChE.Fas2 complex.     

Properties of a Maize Glutathione S-Transferase That Conjugates Coumaric Acid and Other Phenylpropanoids

A glutathione S-transferase (GST) enzyme from corn (Zea mays L. Pioneer hybrid 3906) that is active with p-coumaric acid and other unsaturated phenylpropanoids was purified approximately 97-fold and characterized. The native enzyme appeared to be a monomer with a molecular mass of approximately 30 kD and an apparent isoelectric point at pH 5.2. The enzyme had a pH optimum between 7.5 and 8.0 and apparent Km values of 4.4 and 1.9 mM for reduced glutathione (GSH) and p-coumaric acid, respectively. In addition to p-coumaric acid, the enzyme was also active with o-coumaric acid, m-coumaric acid, trans-cinnamic acid, ferulic acid, and coniferyl alcohol. In addition to GSH, the enzyme could also utilize cysteine as a sulfhydryl source. The enzyme activity measured when GSH and trans-cinnamic acid were used as substrates was enhanced 2.6- and 5.2-fold by the addition of 50 [mu]M p-coumaric acid and 7-hydroxycoumarin, respectively. 1H- and 13C-nuclear magnetic resonance spectroscopic analysis of the conjugate revealed that the enzyme catalyzed the addition of GSH to the olefinic double bond of p-coumaric acid. Based on the high activity and the substrate specificity of this enzyme, it is possible that this enzyme may be involved in the in vivo conjugation of a number of unsaturated phenylpropanoids.