Saul G. Cohen

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.

Effect of solvent in the photoreduction and quenching of benzophenone by triethylamine, 1-azabicyclo[2.2.2]-octane and 1,4-diazabicyclo[2.2.2]octane

Photoreduction of benzophenone by triethylamine (TEA) is efficient in tert-butyl alcohol, acetone and acetonitrile (ϕ = 0.8 – 1.1) and somewhat less efficient in benzene in which evidence of a light-absorbing quenching transient (LAT) is found. Photoreduction by the bridgehead bicyclic amine quinuclidine (1-azabicyclo[2.2.2]octane (ABCO)) is less efficient (ϕ = 0.08 – 0.22) with lower quantum yield in the polar solvents and higher quantum yield in the less polar solvents despite formation of a LAT. Photoreduction by the corresponding bridgehead bicyclic diamine 1,4-diazabicyclo[2.2.2]octane (DABCO) is still less efficient (ϕ = < 0.001 – 0.09) again with lower quantum yield in the polar solvent and the higher in the hydrocarbon. In 1:1 water—pyridine photoreduction of 4-carboxybenzophenone by TEA is efficient (ϕketyl = 1) by ABCO is very inefficient (ϕketyl = 0.0004) and by DABCO leads only to quenching. Low quantum yields for the reduction by ABCO and DABCO in polar solvents are accompanied by high values of the interaction rate constant kir. The results are interpreted in terms of charge transfer interaction between excited triplet carbonyl and non-bonding electrons of the amines. The effects of hyperconjugation and charge delocalization in the charge transfer complex (CTC) and the role of charge transfer complexation and of disproportionation and dissociation to free ions as light-wasting processes are discussed.

Preferred hydrolysis of methyl d-pyroglutamates (5-carbomethoxy-2-pyrrolidones) by α-chymotrypsin

Abstract Methyl d -pyroglutamate, d - III-A , is hydrolyzed by α-chymotrypsin, and the l -enantiomer is not. Methyl α-methylpyroglutamate, III-B , and methyl α-benzylpyroglutamate, III-C , are hydrolyzed by α-chymotrypsin with d -preference based on the glutamic structure, l -preference based on the alanine and phenylalanine structures. γ-Carbomethoxy-γ-valerolactone, IV , is hydrolyzed without stereoselectivity. Kinetics of hydrolysis of the substrates are reported. Modes of association of the substrates with the active site and the relevance of these results to the reactive orientation of the α-acylamido and hydrolyzing groups of noncyclized substrates are discussed.

Triplet-sensitized photobleaching of crystal violet

Abstract Crystal violet (CV + ) and five related triarylmethane dyes quench triplet benzophenone ( 3 K) in acetonitrile at diffusion-controlled rates ( k q ∼ 1 × 10 10 M −1 s −1 ). The interaction with CV + results in bleaching of CV + with a quantum yield, K Φ (−CV + ), dependent on the concentration of CV + with a limiting value of 0.2 at high [CV + . Benzopinacol (K 2 H 2 ) is also formed in this reaction but its yield decreases as [CV + ] increases. In contrast, although both triplet anthracene ( 3 An) and triplet naphthalene ( 3 Nap) are also efficiently quenched by CV + at diffusion-controlled rates, there is only very inefficient fading, An Φ (−CV + ) = 1.1 × 10 −5 . These results are interpreted in terms of a mechanism in which energy transfer to form triplet crystal violet, 3 CV + , is the only major channel in the case of anthracene and naphthalene, i.e. 3 CV + (like 1 CV + ) is unreactive. However, in the case of benzophenone, reduction of 3 K by CV + with a rate constant k h = 1.4 × 10 9 M −1 s −1 to form ketyl radicals (KH . ) can compete (about 14%) with this energy transfer. The ketyl radicals so formed can either reduce CV + to form leuco crystal violet (CVH) or form benzopinacol.

Substituted benzenes and phenols as reversible inhibitors of acetylcholinesterase: Polar, trimethyl, and synergistic effects

Abstract To characterize the nature of reversible binding to acetylcholinesterase of derivatives of benzene and phenol, including the potent inhibitor 3-trimethylammoniophenol, we have carried out a systematic study of effects of substituents on the inhibition by such compounds of the enzymatic hydrolysis of acetylcholine. A linear relation between competitive binding, log 1 K i( com ) , and substituent para -σ values is observed for C 6 H 5 - Y ( Y = HO-, H 2 N-, H-, CH 3 CONH-, CH 3 CO-, and O 2 N-) and for the di-substituted compounds, p -H 2 NCOHN 3 , m -H 2 NNO 2 , p -HONO 2 , m -HONO 2 , and p -O 2 NNHCOCH 3 , with ϱ = +1.84 and correlation, r = 0.99; K i (com) decreases to 0.63 m m for nitrobenzene and 0.46 m m for p -nitroacetanilide. Dimethylamino and trimethylammonio compounds, with substituents (CH 3 ) 2 N-, p -(CH 3 ) 2 NCOCH 3 , m -(CH 3 ) 2 NNO 2 , p -(CH 3 ) 2 NNO 2 and ( fx 237-1, bind more strongly relative to σ values, with ϱ = +2.4 and r = 0.99; K i (com) decreases to 0.09 m m for p -nitrodimethylaniline and phenyltrimethylammonium ion. The observed linear relation of binding energies to substituent σ values indicates that the affinity of these benzene derivatives is determined by electron withdrawal by the substituent and that binding arises from a polar interaction of the benzene ring with an amino acid side chain in an aryl-binding site. The hydroxyl group decreases binding in phenol and the nitrophenols, in accord with its negative σ value. However, consistent with the effect of (CH 3 ) n X-groups in the benzenes, binding of phenols rises linearly with the volume of meta -alkyl and alkylamino substituents in the order, H 3 - 2 H 5 - 3 ) 2 CH- = (CH 3 ) 2 N- 3 ) 3 C-, r = 0.99, with K i values decreasing from 50 m m for phenol to 0.11 m m for 3- tert -butylphenol. This large effect is attributed to binding of the meta -substituent in the trimethyl site, conformational change and synergistic hydrogen bonding of the phenolic hydroxyl. The very strong binding of 3-trimethylammoniophenol K i (com) = 0.00033 m m ) is accurately calculated from the K i of 3- tert -butylphenol, ϱ = 1.84 (above) and the difference in σ values of tert -butyl and trimethylammonio, +0.94. The synergism of (CH 3 ) 3 X- and hydroxyl is the same in the tert -butyl and trimethylammonio phenols. As in trimethylammonio benzene, the positive charge in trimethylammoniophenol has no effect beyond that reflected in its σ value, i.e., in electron withdrawal and in the increase of the polar interaction of the aromatic ring.

Active-site peptides of acetylcholinesterase of Electrophorus electricus: labelling of His-440 by 1-bromo-[2-14C]pinacolone and Ser-200 by tritiated diisopropyl fluorophosphate

To characterize the structure of the active site of acetylcholinesterase (AChE) from the electric organ of E. electricus, we identified sites of incorporation of two active-site affinity labels, [3H]diisopropyl fluorophosphate ([3H]DFP), and 1-bromo-2-[14C]pinacolone ([14C]BrPin). AChE was isolated, purified, inactivated and digested with trypsin, and peptides containing 3H or 14C were purified by reverse-phase HPLC and characterized by N-terminal sequence analysis. [3H]DFP, labelling Ser-200, was found in a single peptide, QVTIFGESAGAASVGMHLLSPDSR, 83% identical with the sequence from Thr-193 to Arg-216 deduced for AChE of T. californica, with Gln, Ala, Leu, and Asp in place of Thr-193, Gly-203, Ile-210 and Gly-214, respectively, and 87% identical with that from bovine and human brain AChEs. Inactivation by [14C]BrPin led to two radioactive peptides. One, ASNLVWPEWMGVIHGYEIEFVFGLPLEK, was 96% identical with that extending from Ala-427 to Lys-454 of T. californica. Release of 14C in cycle 14 established reaction of [14C]BrPin with active-site His-440, protected by 5-trimethylammonio-2-pentanone (TAP). The other peptide, LLXVTENIDDAER, 77% homologous with that of T. californica extending from Leu-531 to Arg-543, had label associated with the third cycle, not protected by TAP, corresponding to Asn-533. The slow inactivation of eel AChE by reaction of [14C]BrPin at His-440 contrasts with that of AChE from T. nobiliana, where it reacts rapidly with a free cysteine, Cys-231, not present in eel AChE. For both AChEs, inactivation by BrPin prevents subsequent reaction with [3H]DFP, and prior inactivation by DFP does not prevent reactions with [14C]BrPin.

EFFECT OF MERCAPTAN ON PHOTOREDUCTION OF ACETONE. NON-REPAIR HYDROGEN TRANSFER REACTIONS†

Abstract— Ultraviolet (u.v.) irradiation of solutions of benzhydrol in acetone leads to formation of ‐2‐propanol, benzpinacol and some benzophenone, apparently from the free radicals (CH3)2COH, II, and (C6H5)2COH, I. 2‐Propanol is formed more rapidly and benzophenone is formed to a much larger extent and persists longer when the solution contains mesityl mercap‐tan, as radical II is reduced by mercaptan and radical I is oxidized by thiyl radical. The same hydrogen atom transfer reactions, which retard by a repair mechanism the photoreduction of benzophenone by 2‐propanol, accelerate and alter the course of photoreduction of acetone by benzhydrol. Irradiation of acetone leads to 2‐propanol, and this is formed more rapidly in the presence of mercaptan. Irradiation of benzophenone in acetone leads to no apparent reaction. The courses of reaction of the several systems are discussed.

Stereoselectivity and reactivity of α-chymotrypsin modified at methionine-192

Abstract Native α-chymotrypsin (N-Chtr) and two modified forms, methionine-192 sulfoxide (O-Chtr) and methionine-192- S -( N -2-carboxyisopropyl)carbamylmethyl (Al-Chtr) α-chy-motrypsin have been compared in hydrolysis of methyl β-phenylpropionate and its d -and l -α-substituted derivatives, in which substituents are Cl, OH, OCOCH 3 , NHCHO, and NHCOCH 3 . In general, both modifications lower the reactivity of the enzymes in terms of k cat and K m (app); the decrease is greater toward l than toward d -enantiomers, and stereoselectivity is decreased. It is proposed that the modifications change the methionine-192 side chain from hydrophobic to hydrophilic, bring it into solution, and enlarge the Met-192-Ser-214 passage in which α-substituents fit. This leads to weaker binding, allows more freedom of motion of substrates and decreases reactivity, while allowing easier access of d -α-substituents. The large modifying substituent of Al-Chtr counters this effect by steric interaction with the large d -α-acetoxy and d -α-acylamido substituents, while the small polar modification in O-Chtr favors hydrolysis of these d -substrates.

Acceleration by aliphatic mercaptan in the photoexcited carbonyl-amine redox system

Abstract Low concentration of aliphatic mercaptan increase the quantum yield of photoreduction of benzophenone by 2-butylamine, nearly to the theoretical maximum value. It is proposed that the effect arises from catalysis by mercaptain of transfer of hydrogen from the amine radical cation to the ketyl radical anion in the charge-transfer of hydrogen complex.

Hydrolysis by Acetylcholinesterase. Trimethyl and Methyl Subsites

A study was made of kinetics of hydrolysis by acetylcholinesterase and by hydroxide of acetate esters RCH2CH2OCOCH3: R = (CH3)3N±, (CH3)3C-, (CH3)2NH±, (CH3)2CH-, CH3 NH±-, CH3CH2-, NH+-, CH3-, H-, HO-, CH30-, CI-, Br-, N=C-. Comparative values of Ks indicate that positive charge of the p-substituent, R, makes little if any contribution to substrate binding. Acylation rate constants, k2, and enzymic reactivities, k2/Ks, are normalized for effects of β-substituents on intrinsic reactivity to hydroxide. A linear relationship is found between log of normalized enzymic reactivity and apparent molal volume of the β-substituent. Cogent evidence is not found for a negative charge in the “anionic” site, which is better considered a Trimethyl site. Enzymic reactivity is determined predominantly by precision of fit of the β-substituent in the Trimethyl site, the acetyl methyl in its Methyl site, and the ester grouping at the serine-OH.