Indophenyl acetate and acetylcholinesterase: binding of a non-specific substrate on the margin of the active center.

1. Indophenyl acetate is a very poor substrate of eel or bovine acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7), with a V less than 5% of that of phenyl acetate, but it is a labile ester and in imidazole buffer is hydrolyzed, non-enzymically, even faster than phenyl acetate. 2. Indophenyl acetate completely protects the enzymes against inhibition by diisopropylphosphorofluoridate but promotes inhibition by methanesulfonyl fluoride. 3. With either of these inhibitors the measured rate of inactivation of eel acetylcholinesterase is the same whether activity is determined with this poor substrate or with a good substrate, acetylthiocholine. With bovine enzyme the inactivation rate is 25% lower when assayed with the former substrate. However this preparation contains a minor enzyme component which is involved in hydrolysis of indophenyl acetate but not good substrates, and which is not readily inhibited. When this is taken into account the inactivation rates for bovine acetylcholinesterase, too, are found to be the same in either assay. These and other observations in the literature can be explained if indophenyl acetate, because of its size, cannot fully penetrate into the active center and is bound in adjoining non-polar regions of the protein. From this external position it makes only intermittent contact with the esteratic site. Hence it is slowly hydrolyzed and fails to protect the enzyme against methanesulfonyl fluoride, though it does protect, possibly sterically, against the larger inhibitor diisopropylphosphorofluoridate.     

Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.     

Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.     

Inactivation of acetylcholinesterase by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride

The neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to reversibly inhibit the activity of acetylcholinesterase. The inactivation of the enzyme was detected by monitoring the accumulation of yellow color produced from the reaction between thiocholine and dithiobisnitrobenzoate ion. The kinetic parameter, K _m for the substrate (acetylthiocholine), was found to be 0.216 mM and K _i for MPTP inactivation of acetylcholinesterase was found to be 2.14 mM. The inactivation of enzyme by MPTP was found to be dose-dependent. It was found that MPTP is neither a substrate of AChE nor the time-dependent inactivator. The studies of reaction kinetics indicate the inactivation of AChE to be a linear mixed-type inhibition. The dilution assays indicate that MPTP is a reversible inhibitor for AChE. These data suggest that once MPTP enters the basal ganglia of the brain, it can inactivate the acetylcholinesterase enzyme and thereby increase the acetylcholine level in the basal ganglia of brain, leading to potential cell dysfunction. It appears that the nigrostriatal toxicity by MPTP leading to Parkinson's disease-like syndrome may, in part, be mediated via the acetylcholinesterase inactivation.     

The mode of binding of potential transition-state analogs to acetylcholinesterase.

Phenylacetone, 4-phenyl-2-butanone, and 4-oxopentyltrimethylammonium chloride were tested as potential transition state analogs for eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7). Phenylacetone is a competitive inhibitor of the enzyme but not a transition state analog, since its binding constant is similar to that for the substrate phenyl acetate. 4-Phenyl-2-butanone binds 6-18 times more tightly than the inhibitors 4-phenyl-2-butanol and N-benzylacetamide and the substrate benzyl acetate and also blocks inactivation of the enzyme with methanesulfonyl fluoride. However, its binding is independent of pH in the range 5-7.5, whereas both V and V/Km for benzyl acetate hydrolysis decrease with decreasing pH in this range. These data indicate a specific but weak interaction between the ketone carbonyl and the enzyme, but probably do not justify considering this compound a transition state analog. 4-oxopentyltrimethylammonium iodide has previously been shown to bind about 125 times more strongly than the substrate acetylcholamine. It also binds about 375 times more strongly than the alcohol 4-hydroxypentyltrimethylammonium iodide. Furthermore, the ketone protects the enzyme from inactivation by methansulfony fluoride, while the corresponding quaternary ammonium alcohol accelerates this inactivation reaction. This additional information confirms that the ketone is a transition state analog.     

Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide

The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.     

Inactivation of gamma-aminobutyric acid aminotransferase by (Z)-4-amino-2-fluorobut-2-enoic acid

(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.     

Fluorinated aldehydes and ketones acting as quasi-substrate inhibitors of acetylcholinesterase.

1. The inhibition of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) by compounds containing trifluoromethyl-carbonyl groups was investigated and related to the effects observed with structurally similar, non-fluorinated chemicals. 2. Compounds that in aqueous solution readily form hydrates inhibit acetylcholinesterase in a time-dependent process. On the other hand non-hydrated, carbonyl-containing compounds showed rapid and reversible, time-independent enzyme inactivation when assayed under steady state conditions. 3. m-N,N,N-Trimethylammonium-acetophenone acts as a rapid and reversible, time-independent, linear competitive inhibitor of acetylcholinesterase (Ki = 5.0 . 10(-7) M). 4. The most potent enzyme inhibitor tested in this series was N,N,N,-trimethylammonium-m-trifluoroacetophenone. It gives time-dependent inhibition and the concentration which inactivates eel acetylcholinesterase to 50% of the original activity after 30 min exposure is 1.3 . 10(-8) M. The bimolecular rate constant for this reaction is 1.8 . 10(6) 1 . mol-1 . min-1. The enzyme-inhibitor complex is very stable as the inhibited enzyme after 8 days of dialysis is reactivated to 20% only. This compound represents a quasi-substrate inhibitor of acetylcholinesterase.     

Kinetic effects of Ca2+ and Mg2+ on ATP hydrolysis by the purified ATP diphosphohydrolase

ATP diphosphohydrolase (EC 3.6.1.5) catalyzes the hydrolysis of diphospho- and triphosphonucleosides and is sensitive to divalent cations. In this paper, we investigated the dependence of ATP hydrolysis on the concentration of free Mg2+ and Ca2+ and the cation ATP complexes. The enzyme was isolated from porcine zymogen granule membranes, solubilized in Triton X-100, and purified on a 5'-AMP-Sepharose 4B affinity column resulting in a 1500-fold purification. Free unprotonated ATP4- was hydrolyzed in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. When hydrolysis rate was measured at different concentrations of the cation-ATP complex at constant free cation concentrations, normal hyperbolic curves were obtained. In CaCl2, both Kapp and Vapp increased as free Ca2+ increased from 25 to 1000 microM. In MgCl2, Kapp increased and Vapp decreased as free Mg2+ increased from 25 to 500 microM. From the rapid equilibrium rate equation, Ks and Vmax values of the substrates were calculated. We found that free ATP4-, Ca-ATP2-, and Mg-ATP2- are substrates and free cations do not bind the enzyme.     

Alcohol dehydrogenase inactivator from rice seedlings : properties and intracellular location

The alcohol dehydrogenase (ADH) inactivator from aerobically grown rice (Oryza sativa) coleoptiles was shown to be associated with membranes which were recovered in sucrose gradients at peak density 1.13 grams per cubic centimeter. When Mg²⁺ was included in the gradient, the inactivator was recovered at peak density 1.16 grams per cubic centimeter coinciding with the marker enzyme for endoplasmic reticulum, antimycin A-insensitive NADH cytochrome c reductase. ADH was recovered exclusively in cytosol fractions. The inactivator attacks ADH from several plant sources and from yeast. There was no evidence for proteolysis when pure yeast ADH was inactivated by the inactivator, but there was a loss of SH groups from ADH during inactivation which was restored after incubation with dithiothreitol under denaturing conditions. The inactivator did not attack other SH enzymes tested but did result in loss of SH groups from glutathione and dithiothreitol which prevent ADH inactivation. When O₂ was removed from the inactivator assay medium, the inactivation as well as the loss of SH groups from yeast ADH was significantly depressed.     

The inactivation of acetylcholinesterase by trimethyloxonium ion, an active-site-directed methylating agent

Trimethyloxonium ion inactivates acetylcholinesterase from the electric eel and acetylcholinesterase on the surface of human red blood cells. Tetramethylammonium ion, which is a competitive inhibitor of acetylcholinesterase, protects against this inactivation. Trimethyloxonium ion does $\text{not}$ inactivate the system that transports choline into the red blood cell. We conclude that trimethyloxonium ion is an affinity-labeling reagent for acetylcholinesterase and that red blood cell acetylcholinesterase is probably not a component of the choline transport system.     

Syntheses of (Z)-and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and their inactivation of gamma-aminobutyric acid aminotransferase.

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respectively) were synthesized and investigated as potential mechanism-based inactivators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core alpha-trifluoromethyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxy-carbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from 4 or 5. This inactivating species is released from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for 4 and 5, respectively. The enzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A mechanism to account for these observations is proposed.     

Purification and spectral studies on the Ca2+-binding properties of 67 kDa calcimedin.

Skeletal-muscle UDP-glucose pyrophosphorylase is inactivated by reaction with 2-ethoxy-N-(ethoxy-carbonyl)-1,2-dihydroquinoline (EEDQ) and 1-(3-dimethylaminopropyl-3-ethylcarbodi-imide (EDAC), two reagents specific for carboxylate groups. The former reagent is a more effective inactivator than EDAC. Although no evidence of reversible enzyme-reagent complexes of the affinity-labelling type was obtained by kinetic analysis of the inactivation, the selective protection of UDP-glucose pyrophosphorylase activity against inactivation by EEDQ in the presence of uridine substrates is indicative of an active-site-directed effect. The results are consistent with the hypothesis that EEDQ modifies a single carboxylate group located in a hydrophobic domain close to the substrate-binding site, leading to enzyme inactivation. In contrast, the reaction between UDP-glucose pyrophosphorylase and EDAC appears to involve a different region of the enzyme.     

Mechanism of inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone

The mechanism of inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone has been studied. Chloride analysis of the inactivated enzyme suggests that the complex does not contain intact chloropyrone or an acid chloride. 13C NMR studies of the enzyme inactivated with 13C-enriched chloropyrones show that (1) the pyrone ring is no longer intact, (2) C-6 becomes a carboxylate group and C-2 becomes esterified to the enzyme, probably to serine-195, and (3) a double bond is present adjacent to the serine ester. The inactivated enzyme slowly regains catalytic activity with the concomitant release of (E)-4-benzyl-2-pentenedioic acid. It is concluded that double bond migration occurs during reactivation since the position of the double bond in the released diacid product is different than in the inactivator-enzyme complex. When the reactivation is carried out in [18O]H2O-enriched water, a single oxygen-18 is incorporated into the released product and is further evidence that the inactivator is bound to the enzyme only through a single ester linkage. A deuterium isotope effect on reactivation is observed when a chloropyrone deuterated at C-5 is used. This result demonstrates that removal of a proton from C-5 is required for reactivation and that isomerization of the double bond and not hydrolysis of the acyl-enzyme is rate determining. A variety of amines accelerate the rate of reactivation by functioning as general bases and not as nucleophiles. A reaction scheme is presented that accounts for the formation of the stable inactivator-enzyme complex as well as the production of two products derived from enzymatic hydrolysis of the chloropyrone.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cystine-dependent inactivator of tyrosine aminotransferase co-purifies with gamma-cystathionase (cystine desulfurase)

Tyrosine aminotransferase is stable in homogenates of rat liver, but not when L-cystine or L-cysteine is added, which causes the enzyme to be reversibly inactivated due to oxidation of thiol groups. By monitoring inactivation of the aminotransferase in the presence of L-cystine, a factor responsible for this loss of activity was purified from rat liver. The factor required vitamin B6 and co-purified with gamma-cystathionase during numerous steps. Highly purified inactivating factor contained a protein that was identical in size and isoelectric point to cystathionase but also contained a dissimilar peptide that appeared to be unrelated to cystathionase. Cystathionase and the cystine-dependent inactivator shared several catalytic activities, including the hydrolysis of cystathionine, desulfuration of cystine, and desulfhydration of cysteine. During incubation of L-cysteine with the purified factor, hydrogen sulfide was generated but no inactivation of the aminotransferase occurred, suggesting that cysteine-dependent inactivation requires additional mechanisms. An insoluble inactivator of tyrosine aminotransferase that is produced during the reaction may be elemental sulfur, since colloidal suspensions of sulfur also inhibited the enzyme. Another inhibitor fractionated with high molecular weight substances; this may be protein-bound sulfane.     

Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide

The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.Abbreviations ALP alkaline phosphatase - PNPP p-nitrophenyl phosphate - NBS N-bromosuccinimide     

Alcohol dehydrogenase and an inactivator from rice seedlings

Alcohol dehydrogenase (ADH) was measured in the various organs of rice seedlings (Oryza sativa) growing in air. In extracts from ungerminated seeds, the ADH is stable, but in extracts from seedlings more than 2 days old the enzyme initially present loses activity in a time- and temperature-dependent fashion, due to the presence of an inactivating component which increases with age in roots and shoots. The inactivation can be prevented completely by dithiothreitol, and when this is included in the extraction medium the apparent loss of total ADH in roots and shoots with age is not observed. In seedlings grown in N₂, ADH levels in coleoptile extracts are higher than those in air, the enzyme is stable, and no inactivator can be detected. When seedlings grown for 5 days in air were transferred to N₂ for 3 days, ADH levels increased and there was a decline in inactivator activity. Transfer back to air after 1 day in N₂ led to loss of the accumulated ADH and increase in inactivator. These reciprocal changes and the fact that the inactivator is absent from coleoptiles of seedlings grown in N₂ appear to suggest a regulatory role for the inactivator in vivo. However, it is clear that high levels of inactivator and ADH can exist in cells of seedlings grown in air for long periods without loss of enzyme activity, and it is argued that they must normally be separately compartmented.     

Interaction of beta-iodopenicillanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39.

The beta-lactamases of Streptomyces albus G and Actinomadura R39 are inactivated by beta-iodopenicillanate. However, in contrast with the beta-lactamase I from Bacillus cereus, they also efficiently catalyse the hydrolysis of the inactivator; with the S. albus G enzyme, kcat. is larger than 25s-1 and the number of turnovers before inactivation is 515. With the A. R39 enzyme, kcat. is larger than 50s-1 and the number of turnovers before inactivation is 80. After hydrolysis of the beta-lactam amide bond, the product rearranges into 2.3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which exhibits an absorption maximum at 305 nm.     

pH effects in the spontaneous reactivation of phosphinylated acetylcholinesterase

Previous studies on the spontaneous reactivation of phosphorylated and phosphonylated cholinesterases report bell-shaped curves with reaction rate maxima between pH values of 7 and 9. By way of contrast, we found reactivation rate minima in the same pH region for a phosphinylated bovine erythrocyte acetylcholinesterase and three phosphinylated eel acetylcholinesterases. To further elucidate these observations, eel acetylcholinesterase was inhibited with racemic 4-nitrophenyl ethyl(phenyl)phosphinate. The spontaneous reactivation of the inhibited enzyme over the pH range 6.00 to 9.00 was monitored following 1. both inhibition and spontaneous reactivation at the same pH, and 2. inhibition at pH 7.60 followed by spontaneous reactivation at the selected pH. The combined plots of both studies gave overlapping pH curves with minima around pH 7.60. The results indicate that the minima in the rates of the spontaneous reactivation of phosphinylated acetylcholinesterases are not the consequence of a pH-controlled change in the relative inhibition rates of the P(+)- and P(-)-enantiomers participating in the inhibition reaction. Our results suggest that the orientation of the phosphinyl group in the active site of phosphinylated acetylcholinesterase is quite different from that of the inhibitor groups in phosphonylated or phosphorylated enzyme.     

Kinetics of modification of the mitochondrial succinate-ubiquinone reductase by 5,5′-dithiobis-(2-nitro-benzoic acid)

The kinetic theory of the substrate reaction during modification of enzyme activity previously described by Tsou [Tsou (1988),Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381–436] has been applied to a study of the kinetics of the course of inactivation of the mitochondrial succinate-ubiquinone reductase by 5,5′-dithiobis-(2-nitro-benzoic acid) (DTNB). The results show that the inactivation of this enzyme by DTNB is a conformation-change-type inhibition which involves a conformational change of the enzyme before inactivation. The microscopic rate constants were determined for the reaction of the inactivator with the enzyme. The presence of the substrate provides marked protection of this enzyme against inactivation by DTNB. The modification reaction of the enzyme using DTNB was shown to follow a triphasic course by following the absorption at 412 nm. Among these reactive thiol groups, the fast-reaction thiol group is essential for the enzyme activity. The results suggest that the essential thiol group is situated at the succinate-binding site of the mitochondrial succinate-ubiquinone reductase.