Several peptides of acetylcholinesterase of Torpedo californica labelled with the alkylating reagent [3H]N,N‐dimethyl‐2‐phenyl‐aziridinium (DPA) were localized within the primary structure. One peptide had the sequence KPQELIDVE (positions 270‐278); the incorporation of DPA into this peptide could be specifically suppressed by propidium, which suggests that it is part of the peripheral anionic site. The incorporation of DPA into two other peptides was insensitive to propidium but could be prevented by edrophonium; the sequence of one of the peptides assumed to be part of the anionic site in the catalytic centre was found to be DLFR (positions 217‐220). Decamethonium efficiently blocked alkylation by DPA in all three investigated peptides.

The Michaelis-Menten parameters k,,,, Ks(app) and the second-order rate constants kll = kz/K, of acetylcholinesterase-catalyzed hydrolysis of 25 acetic esters with non-ionic leaving groups have been determined at 25 "C and pH 7.5 in 0.1 5 M KCI. A linear relationship between the substrate noncovalent binding capacity and the leaving group hydrophobicity, and a multiparameter correlation of the acetylation reaction rate constant logarithm with the leaving group inductive effect, hydrophobicity, and steric effect, have been established. The acetyl-enzyme deacetylation rate constant has been calculated. Taken together, a fairly complete understanding of acetylcholinesterase specificity is possible.The data are consistent with a model of the acetylcholinesterase active site, in which the catalytically active groups are located at the bottom of a jaws-like slit with a limited range of hydrophobic walls that provide the sorption of the substrate leaving groups not longer than that in n-butyl acetate.Acetylcholinesterase has been shown to hydrolyse, in addition to its specific substrate acetylcholine, many other acetic esters [l]. This provides a prospect for extensive study of the influence of substrate leaving group structure on the effectiveness of acetylcholinesterase-catalyzed hydrolysis. A conclusion that both the substituent inductive effect and hydrophobicity are important in acetylcholinesterase reactions, can be drawn from the results of previous studies [2-121. In this paper we are concerned with quantitative evaluation of these influences in the reaction series CH3C-(O)OX, where the group X includes hydrocarbon chains and various non-ionic electronegative substituents (see Table 1).Up to now the analysis of enzyme kinetics data by means of correlation equations has been carried out mainly on chymotrypsin-catalyzed reactions [13 -161. In accordance with the three-step reaction scheme(2) (3) the structure-activity relationship for the non-covalent binding step (3) has been given by the equation where K, = k-l/kl, while E is enzyme, S substrate, ES the enzyme-substrate complex, EA the acyl-enzyme, PI and P2 the first and second products. For the acylation reaction (2), as well as for the deacylation (3), the multiparameter correlation equation log k2 = log k2" + @* O* + van + 6 E, ( 5 ) can be applied. The parameters e*, q and 6 in the equations are the intensity factors of the inductive effect, hydrophobicity and steric effect, respectively. The constants cr* and E, have their usual meanings as in physical organic chemistry [17]. The hydrophobicity constants n have been introduced by Hansch [18,19].Under the conditions where the product and substrate inhibition phenomena could be left out of consideration, the reaction sequence (1 -3) would be applicable to the acetylcholinesterase-catalyzed hydrolysis of esters [20]. According to the reaction scheme the meaning of the constants kcat and Ks(app) (K, = K, if k2 < k-l) in the Michaelis-Menten equation depends on the values of the ratio kz/kj as follows : (7)

No abstract

Acetylcholinesterase from cobra (Naja naja oxiana) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate. Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67000 ± 2000. At high enzyme concentrations (> 0.2 mg/ml, > 1 μM) and ionic strength 0.1 M, it reversibly tends to form higher‐molecular‐weight 7.1‐S aggregates. Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.

A peptide of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) from the venom of the cobra Naja naja oxiana labeled by the affinity reagent N,N-dimethyl-2-phenylaziridinium (DPA) has been identified. The sequence is Gly-Ala-Glu-Met-Trp-Asn-Pro-Asn. In AcChoEase from Torpedo californica, a homologous peptide was labeled and isolated. Its sequence is Ser-Gly-Ser-Glu-Met-Trp-Asn-Pro-Asn, representing positions 79 through 87. In both cases labeling can be prevented by 0.1 mM edrophonium, indicating that the respective peptides form part of the anionic subsite of the catalytic center. The modified residue was tryptophan (Trp-84 in Torpedo AcChoEase) in both enzymes. In contrast to AcChoEase from Torpedo, the enzyme from cobra venom does not contain a peripheral anionic binding site.

Nucleophilic efficiency of free amino acids in chymotrypsin-catalyzed acyl transfer in ice at -18°C using ethyl esters of N-maleyl-r.-tyrosine and L-tyrosine as the acyl group donors has been studied. Although the amino acids did not act as acyl acceptors in liquid water, the high yields of peptides were obtained in frozen solutions at pH 10.5 (before freezing). The efficiency of amino acids in the formation of the corresponding dipeptides depended on the substrate used, and decreased in the order Ser,Thr,Gln>Lys>Cit>Ala>Gly>Asn>Arg>Glu>V~>Om>Asp (with no peptide formed with His, Leu, Be and Pro) for N-maleyl-L-tyrosine ethyl ester and Ser>Lys>Om>Arg,Cit>Gln>Thr>Asn>Ala>Gly (with no peptide formed with Glu, Val, Asp, His, Leu, Be and Pro) for L-tyrosine ethyl ester. a-Chymotrypsin; Nucleophile specificity; Frozen solution

No abstract

No abstract