Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities

The effect of chemical modification on the acetylcholinesterase and the aryl acylamidase activities of purified acetylcholinesterase from electric eel and basal ganglia was investigated in the presence and absence of acetylcholine, the substrate of acetylcholinesterase, and 1,5-bis[4-(allyldimethylammonium)phenyl]pentan-3-one dibromide (BW284C51), a reversible competitive inhibitor of acetylcholinesterase. Trinitrobenzenesulfonic acid, pyridoxal phosphate, acetic anhydride, diethyl pyrocarbonate, and 2-hydroxy-5-nitrobenzyl bromide under specified conditions inactivated both acetylcholinesterase and aryl acylamidase in the absence of acetylcholine and BW284C51. Chemical modifications in the presence of acetylcholine and BW284C51 by all the above except diethyl pyrocarbonate selectively prevented the loss of acetylcholinesterase but not aryl acylamidase activity; modification by diethyl pyrocarbonate in the presence of acetylcholine and BW284C51 prevented the loss of both acetylcholinesterase and aryl acylamidase activities. Treatment with N-acetylimidazole resulted in the inactivation of acetylcholinesterase and the activation of aryl acylamidase. These changes in both the activities could be prevented by acetylcholine and BW284C51. Modification by phenylglyoxal, 2,4-pentanedione, or N-ethylmaleimide did not affect the enzyme activities. Indophenylacetate hydrolase activity followed a pattern similar to that of acetylcholinesterase in all the above modification studies. The results suggested essential lysine, tyrosine, tryptophan, and histidine residues for the active center of acetylcholinesterase and essential lysine, histidine, and tryptophan residues for the active center of aryl acylamidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification studies of Rhizomucor miehei protease: evidence for the role of basic amino acids in enzyme catalysis.

The effect of chemical modification on milk clotting and proteolytic activities of aspartyl protease obtained from Rhizomucor miehei NRRL 3500 was examined in the absence and the presence of its specific inhibitor pepstatin A. The effect on the ratio of milk clotting activity (MC) to proteolytic activity (PA), an index of the quality of milk clotting proteases was also determined. Modification of the enzyme with trinitrobenzenesulfonic acid, diethylpyrocarbonate and phenylglyoxal produced an increase in the ratio of MC/PA, while modification with 2- hydroxy-5-nitrobenzyl bromide did not affect the ratio. Modification with N-acetylimidazole resulted in a marginal increase in MC/PA ratio. Protection using pepstatin A during modification with phenylglyoxal, N-acetylimidazole and 2-hydroxy-5-nitrobenzyl bromide, protected both MC and PA. In the case of modification by diethylpyrocarbonate, pepstatin A protected only MC. Pepstatin A did not protect both the activities on the modification of the enzyme by trinitrobenzene sulfonic acid. These observations indicate the presence of arginine, tyrosine and tryptophan at the catalytic site of the enzyme, for eliciting MC and PA of the enzyme. In general, modification of the positively charged residues increases the MC/PA ratio of the enzyme. In addition the modified lysine residues responsible for the inactivation of the enzyme were not involved in the active site of the enzyme. Thus the lysine residues might have a secondary role in enzyme catalysis. Further, histidine at the catalytic site was found to be exclusively involved in milk clotting activity. The enzyme with modified histidine residues were more susceptible to autocatalysis, indicating that histidine residues protect the enzyme against autolysis.     

Amino acid residues essential for catalysis by peptidyl dipeptidase-4 from Pseudomonas maltophilia

To assess residues essential for catalysis by prokaryotic peptidyl dipeptidase-4, the enzyme was subjected to chemical modification by a series of reagents. Treatment with either tetranitromethane or N-acetylimidazole abolished catalytic activity. Hydroxylamine reversed inactivation by acetylimidazole only. Thus, an essential tyrosine is indicated. Enzymatic activity also was quenched by either trinitrobenzenesulfonic acid or diethyl pyrocarbonate. Inactivation by these reagents was not reversed by hydroxylamine. These data suggest an essential lysine. The competitive inhibitor Phe-Arg protected partially against inactivation by tetranitromethane, and fully against inactivation by N-acetylimidazole. The substrate Hip-Phe-Arg protected against inactivation by trinitrobenzenesulfonic acid and diethyl pyrocarbonate. Thus, both tyrosine and lysine are located at the catalytic site.     

Purification and active site modification studies on glyoxalase I from monkey intestinal mucosa

Glyoxalase I ((R)-S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from monkey intestinal mucosa was purified to homogeneity. The purified enzyme had a molecular weight of 48,000, composed of two apparently identical subunits. Active-site modification was carried out on the purified enzyme in presence and absence of S-hexylglutathione, a reversible competitive inhibitor of glyoxalase I. Modification by tetranitromethane and N-acetylimidazole caused inactivation of the enzyme. Inactivation by N-acetylimidazole was reversible with hydroxylamine treatment, suggesting the importance of tyrosine residues for the activity of the enzyme. The enzyme was inactivated by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, 2,4,6-trinitrobenzenesulphonic acid, pyridoxal phosphate and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, indicating the importance of tryptophan, lysine and glutamic acid/aspartic acid residues for the activity of the enzyme. The enzyme was inactivated by diethyl pyrocarbonate and the activity was not restored by hydroxylamine treatment, suggesting that histidine residues may not be important for activity. Modification by N-ethylmaleimide and p-hydroxymercuribenzoate did not affect its activity, indicating that sulphydryl groups may not be important for activity. These studies indicated that the amino acids present in the active site of glyoxalase I from intestinal mucosa which may be important for activity are tyrosine, tryptophan, lysine and glutamic acid/aspartic acid residues.     

Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.     

Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker''s yeast).

The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.     

Role of tryptophan, histidine and methionine residues in the catalytic activity of mitochondrial aspartate aminotransferase from beef kidney.

The role of tryptophan, methionine, and histidine residues in mitochondrial aspartate aminotransferase from beef kidney has been established by using N-bromosuccinimide, 2-hydroxy-5-nitrobenzylbromide, and tetraiodofluoresceine as specific chemical modifiers of the amino acid residues of the enzyme. Since N-bromosuccinimide promotes extensive inactivation of the enzyme and the chemical modification of 1.65 tryptophan and 3 methionine residues per enzymes protomer, 2-hydroxy-5-nitrobenzylbromide modifies once more 1.65 tryptophan residues per enzyme protomer but induces only 10% inactivation of the enzyme. Tetraiodofluoresceine exerts a 40% inactivation of the enzyme which is due to the chemical modification of 5.8 histidine res in     

Characterization of the amino acids of bovine fibrinogen involved in the fibrinogen-thrombin interaction of the blood clotting process. Comparison with the milk clotting process

Summary Bovine fibrinogen and the A and B chains of bovine fibrinogen have been subjected to chemical modification by a number of reagents and the effects of these procedures on the susceptibility of the proteins to thrombin hydrolysis is described. The reagents used were rose bengal (for photo-oxidation), 2-hydroxy-5-nitrobenzyl bromide, N-acetylimidazole, iodoacetic acid and diethyl pyrocarbonate. Evidence is presented which indicates that the tryptophan and tyrosine residues of fibrinogen are not involved to any great extent in the interaction of this protein with thrombin. Modification with iodoacetic acid suggests that methionine residues play a major role in such interactions, but the fibrinogen chains on which the important residues reside remain uncertain. The use of diethyl pyrocarbonate indicates the participation also of histidine in fibrinogen-thrombin interactions and that, whereas the histidine residues of the B chain are involved to a great extent, it appears that those of the Aa chain are not. The similarities which exist between the fibrinogen-thrombin and the -casein-chymosin systems are discussed.Abbreviations used DEP diethyl pyrocarbonate (ethoxyformic anhydride) - HNBB 2-hydroxy-5-nitrobenzyl bromide - N-Acl N-acetylimidazole - PTC phenylthiocarbamyl - PTH 3-phenyl-2-thiohydantoin.     

Effect of prior treatment with Clostridium perfringens epsilon toxin inactivated by various agents on lethal, pressor and contractile activities of the toxin

Lethal and pressor activities, and the contractile responses of rat isolated ileum to Clostridium perfringens epsilon toxin, were significantly prevented by the prior administration of epsilon toxin inactivated by 1-ethyl-3-(3-diethyl-aminopropyl) carbodiimide in the presence of glycine methyl ester (EDC), 2,4,6-trinitrobenzene sulfonic acid (TNBS), succinic anhydride (SA) and ethoxyformic anhydride (EFA). However, the prior administration of the toxin inactivated by N-acetylimidazole (NAI), tetranitromethane (TNM) and N-bromosuccinimide (NBS) resulted in no inhibition of these biological activities. These data suggest that the toxin interacts with specific site(s) on target organs or tissues. The relationship between amino acid residues and the actions of the toxin is described.     

Chemical modification of rat liver arginase

Chemical modifications were used to search for catalytically important residues of rat liver arginase. The results of carbamoylation, nitration and diazotization suggest that lysyl and tyrosyl residues are not involved in the catalytic function of arginase. The modification of 5--6 tryptophanyl residues by N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide led to about 90% inhibition of the enzyme activity. Photooxidation of 21 histydyl residues also led to considerable inactivation of arginase. The modification of tryptophanyl and histidyl residues did not cause dissociation of the enzyme into subunits.     

Chemical modification studies on a lectin from winged-bean [Psophocarpus tetragonolobus (L.) DC] tubers.

The effect of chemical modification on a D(+)-galactose-specific lectin isolated from winged-bean tubers was investigated to identify the type of amino acid involved in its haemagglutinating activity. Various anhydrides of dicarboxylic acids, such as acetic anhydride, succinic anhydride, maleic anhydride and citraconic anhydride, modified 57-68% of the amino groups of the winged-bean tuber lectin. Treatment with N-acetylimidazole modified only 45% of the total amino groups. Reductive methylation of free amino groups modified 57% of the amino groups. Modification of the amino groups of the lectin by acetic anhydride and succinic anhydride did not lead to any significant change in the haemagglutinating activity (greater than or equal to 75% active). However, citraconylation and maleylation of the lectin led to a significant decrease in the haemagglutinating activity (less than or equal to 20% active). Acetylation and succinylation (3-carboxypropionylation) of the lectin led to a decrease in the pI value of the native lectin from approx. 9.5 to approx. 4.5. Treatment of the lectin with N-bromosuccinimide led to the modification of two and four tryptophan residues per molecule in the absence and in the presence of 8 M-urea respectively. The immunological identity of all the modified lectin preparations showed no gross structural changes except the lectin modified with N-bromosuccinimide in the presence of urea at pH 4.0.     

Impairment by covalent modification of the ability of Phaseolus vulgaris phytohemagglutinin to stimulate cultured human lymphocytes: a comparison of different forms of covalent modification

Phytohemagglutinin (PHA) isolated from Phaseolus vulgaris has been modified by treatment with various chemical reagents and the modified proteins have been tested for their ability to stimulate peripheral lymphocytes from two healthy human donors, in vitro. Reaction of PHA with citraconic anhydride, S-methyl isothiourea, or 2-hydroxy-5-nitrobenzyl bromide produced derivatives which retained the ability to stimulate lymphocytes, at low concentrations. Acylation of the lectin with acetic anhydride or masking of the carboxyl side chains by reaction with glycinamide-carbodiimide impaired stimulation. When PHA was treated with N-bromosuccinimide or with tetranitromethane, the derivatives were ineffective as lymphocyte stimulants. Chemical modifications affected, in some cases, the quaternary structure of the lectin. Glycinamide-, homoarginine-, and nitro-PHA were tetramers whereas acetyl-, citraconyl-, and N-bromosuccinimide-treated lectin were dimers. Antinative lectin antiserum cross-reacted with all the modified proteins, except in the case of the N-bromosuccinimide derivative. The results show that, in the human lymphocyte transformation assay, the mitogenic property of PHA may depend on intact aspartic, glutamic, and tyrosine residues whereas lysine residues do not appear to be essential.     

Identification of serotonin-sensitive aryl acylamidase activity with cobra venom acetylcholinesterase

Acetylcholinesterase purified from cobra (Naja naja) venom exhibits a serotonin-sensitive aryl acylamidase activity. Both acetylcholinesterase and aryl acylamidase activities co-eluted in column chromatographic procedures (Sephadex G-75 and Zinc-Sepharose), co-migrated on polyacrylamide gel electrophoresis, co-immunoprecipitated by anti-snake venom antibody and showed the same heat denaturation profile at 40 degrees C. Further, several potent acetylcholinesterase inhibitors at different concentrations inhibited the cholinesterase and aryl acylamidase activities to the same extent. It is concluded that in cobra venom, acetylcholinesterase is associated with a serotonin-sensitive aryl acylamidase activity similar to earlier observations made with acetylcholinesterase from different sources.     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.     

Essential residues in angiotensin converting enzyme: modification with 1-fluoro-2,4-dinitrobenzene

The peptidase and esterase activities of rabbit pulmonary angiotensin converting enzyme (ACE) are rapidly abolished on reaction with 1-fluoro-2,4-dinitrobenzene (Dnp-F). Inactivation follows first-order kinetics with respect to the reagent and is accompanied by stoichiometric incorporation of 3,5-[3H]Dnp, indicating that the effect is due to a specific modification of the enzyme. Thin-layer chromatography of an acid hydrolysate of the modified enzyme indicates that most of the radioactive label is present as O-Dnp-tyrosine (65 to greater than 95%) and the rest as N epsilon-Dnp-lysine. The pH dependence of the reaction is consistent with modification of either tyrosine or lysine. The presence of a competitive inhibitor effectively protects the enzyme against inactivation by Dnp-F. Acetylation of ACE with N-acetylimidazole also protects the enzyme against modification with Dnp-F. The results indicate the presence of catalytically essential tyrosine and lysine residues at the active site of ACE.     

Chemical modification of histidine, tyrosine, tryptophan and cysteine residues in carp (Cyprinus carpio) muscle enolase

Enolase from carp (Cyprinus Carpio) muscle was modified by diethylpyrocarbonate, tetranitromethane, N-bromosuccinimide and 5,5'-dithiobis(2-nitrobenzoic acid). The extent and rate of modification and its effect on the enzyme activity were determined. Modification of histidine, tyrosine and tryptophan residues caused complete inactivation of the enzyme; Mg2+ as well as 2-phosphoglycerate markedly altered the rates of modification and inactivation. The above-mentioned amino acid residues seem to be essential for the functioning of muscle enolases. Modification of cysteine residues had no effect on the enolase activity.     

The chemical modification of the essential groups of beta-N-acetyl-D-glucosaminidase from Turbo cornutus Solander

The chemical modification of beta-N-acetyl-D-glucosaminidase (EC3.2.1.30) from Turbo cornutus Solander has been first studied. The results demonstrate that the sulfhydryl group of cysteine residues and the hydroxyl group of serine residues are not essential to the enzyme's function. The modification of indole group of tryptophan of the enzyme by N-bromosuccinimide (NBS) can lead to the complete inactivation, accompanying the absorption decreasing at 278 nm and the fluorescence intensity quenching at 335 nm, indicating that tryptophan is essential residue to the enzyme. The modification of amino group of lysine residue by formaldehyde and trinitrobenzenesulfonic acid also inactivates the enzyme completely. The results show that lysine and tryptophan are probably situated in the active site of the enzyme. The modification of the imidazole residue and carboxyl group leads to inactivate incompletely, indicating they are not the composing groups of the enzyme active center, and they are essential for maintaining the enzyme's conformation which is necessary for the catalytic activity of the enzyme.     

Acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase. Evidence for an active site histidine residue

The lysosomal membrane enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase catalyzes the transfer of the acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction appears to be a transmembrane process: the enzyme is acetylated on the outside of the lysosome, and the acetyl group is transferred across the membrane to the inside of the lysosome where it is used to acetylate glucosamine. To determine the reactive site residues involved in the acetylation reaction, lysosomal membranes were treated with various amino acid modification reagents and assayed for enzyme activity. Although four thiol modification reagents were examined, only one, p-chloromercuribenzoate inactivated the N-acetyltransferase. Thiol modification by p-chloromercuribenzoate did not appear to occur at the active site since inactivation was still observed in the presence of the substrate acetyl-CoA. N-Acetyltransferase could be inactivated by N-bromosuccinimide, even after pretreatment with reagents specific for tyrosine and tryptophan, suggesting that the modified residue is a histidine. Diethyl pyrocarbonate, another histidine modification reagent, could also inactivate the enzyme; this inactivation could be reversed by incubation with hydroxylamine. N-Bromosuccinimide and diethyl pyrocarbonate modifications appear to be at the active site of the enzyme since co-incubation with acetyl-CoA protects the N-acetyltransferase from inactivation. This protection is lost if glucosamine is also present. Pre-acetylated lysosomal membranes are also able to provide protection from N-bromosuccinimide inactivation, providing further evidence for a histidine moiety at the active site and for the existence of an acetyl-enzyme intermediate.     

Chemical modification of liquefying alpha-amylase: role of tyrosine residues at its active center

Liquefying alpha-amylase from Bacillus amyloliquefaciens was inactivated by treatment with tetranitromethane and N-acetylimidazole. The loss of activity occurred with modification of five tyrosine residues. Preincubation of the enzyme with either the substrate or the competitive inhibitor at saturating levels provided complete protection against inactivation. However, the presence of substrate/inhibitor in the reaction mixture protected only two of the five modifiable tyrosine residues, suggesting the involvement of only two tyrosine residues at the active center. This was confirmed when hydroxylamine treatment of the acetylated enzyme fully restored the enzymatic activity. Both nitration and acetylation increased the apparent Km of the enzyme for soluble starch, which indicated that the tyrosine residues are involved in substrate binding. Reduction of nitrotyrosine residues to aminotyrosine residues failed to restore the enzymatic activity. So, the loss of activity on modification of tyrosine residues was ascribed to conformational perturbances and not simply to the changes in the ionic character of tyrosine residues.     

Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.