REACTION OF ENZYMES AND PROTEINS WITH MUSTARD GAS (BIS(β-CHLOROETHYL)SULFIDE)

1. The rate of reaction of mustard gas (H) with thirteen proteins has been determined. The extreme variation in reaction rates is about 100:1. 2. No qualitative difference in the results was observed when the treatment with H was carried out by the Dixon or stirring methods. 3. The kinetics have been analyzed and a bimolecular equation derived which fits the facts. 4. The carboxyl groups of all proteins reacted when the reaction with H was carried out at pH 6.0 in M/25 acetate buffer. In most cases the number of carboxyl groups covered was approximately equal to the number of H residues bound. 5. The amino groups of proteins failed to react with the possible exception of yeast hexokinase. 6. The color obtained when proteins were mixed with Folin''s phenol reagent at pH 8.0 decreased as the protein was treated with H. The color returned on treatment of the H-protein with alkali and many of the combined H groups were hydrolyzed. Similar results were observed when a concentrated glycyltyrosine solution was treated with H.     

THE REACTIONS OF IODINE AND IODOACETAMIDE WITH NATIVE EGG ALBUMIN

The following experimental results have been obtained. 1. Native egg albumin treated with iodine and then denatured no longer gives a nitroprusside test or reduces dilute ferricyanide in neutral Duponol PC solution. 2. More iodine is needed to abolish the ferricyanide reduction if the reaction between native egg albumin and iodine is carried out at pH 6.8 than if the reaction is carried out at pH 3.2. At pH 6.8 iodine reacts with tyrosine as well as with cysteine. 3. Cysteine and tryptophane are the only amino acids with reducing groups which are known to react with dilute iodine at pH 3.2 The reducing power of cysteine is abolished by the reaction with iodine, whereas the reducing power of tryptophane remains intact. Pepsin and chymotrypsinogen which contain tryptophane but not cysteine, do not react at all with dilute iodine at pH 3.2. 4. Native egg albumin treated with iodoacetamide at pH 9.0 and then denatured by Duponol PC reduces only 60 per cent as much dilute ferricyanide as egg albumin which has not been treated with iodoacetamide. 5. The SH group is the only protein reducing group which is known to react with iodoacetamide. The simplest explanation of the new observation that the SH groups of egg albumin can be modified by reactions with the native form of the protein is that the native egg albumin has free and accessible but relatively unreactive SH groups which can react with iodine and iodoacetamide despite the fact that they do not react with ferricyanide, porphyrindin, or nitroprusside. Preliminary experiments suggested by the results with egg albumin indicate that the tobacco mosaic virus is modified by iodine at pH 2.8 without being inactivated and that the tobacco mosaic and rabbit papilloma viruses are not inactivated by iodoacetamide at pH 8.0.     

Modification of arginine residues in calf intestinal alkaline phosphatase

Calf intestinal alkaline phosphatase is inactivated by 2,3-butanedione and phenylglyoxal. The reaction with either reagent results in a biphasic loss of enzymatic activity. Inactivation by 2,3-butanedione in borate buffer can be reversed after gel-filtration in Tris buffer but no enzyme reactivation is observed after phenylglyoxal treatment. Phosphate, ATP and NADH protect the enzyme from both compounds while no protection is displayed by L-phenylalanine. The selective chemical modification indicates that two differently reacting types of arginines are present in the active site domains of the dimeric enzyme.     

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: fluorescent affinity labeling of the catalytic subunit from bovine skeletal muscle with o-phthalaldehyde

The catalytic subunit of adenosine cyclic 3',5'-monophosphate dependent protein kinase from bovine skeletal muscle was rapidly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics, and the second-order rate constant was 1.1 X 10(2) M-1 s-1. Absorbance and fluorescence spectroscopic data were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme). The reaction between the catalytic subunit and o-phthalaldehyde was not reversed by the addition of reagents containing free primary amino and sulfhydryl functions following inactivation. The reaction, however, could be arrested at any stage during its progress by the addition of an excess of cysteine or less efficiently by homocysteine or glutathione. The catalytic subunit was protected from inactivation by the presence of the substrates magnesium adenosine triphosphate and an acceptor serine peptide substrate. The decrease in fluorescence emission intensity of incubation mixtures containing iodoacetamide- or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified catalytic subunit and o-phthalaldehyde paralleled the loss of phosphotransferase activity. Catalytic subunit denatured with urea failed to react with o-phthalaldehyde. Inactivation of the catalytic subunit by o-phthalaldehyde is probably due to the concomitant modification of lysine-72 and cysteine-199. The proximal distance between the epsilon-amino function of the lysine and the sulfhydryl group of the cysteine residues involved in isoindole formation in the native enzyme is estimated to be approximately 3 A. The molar transition energy of the catalytic subunit-o-phthalaldehyde adduct was 121 kJ/mol and compares favorably with a value of 127 kJ/mol for the 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl)isoindole in hexane, indicating that the active site lysine and cysteine residues involved in formation of the isoindole derivative of the catalytic subunit are located in a hydrophobic environment. o-Phthalaldehyde probably acts as an active site specific reagent for the catalytic subunit.     

Identification of subsidiary catalytic groups at the active site of beta-ketoacyl-CoA thiolase by covalent modification of the protein

beta-Ketoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) is known to possess sulfhydryl groups of cysteines at the active site that are essential for its catalytic activity. Other groups at the active site that participate in the catalytic process were identified by using anhydride reagents which covalently modify the protein by specifically reacting with any amino groups potentially present at the active site. Since these reagents may also react with thiol groups, the enzyme's amino groups were modified after masking the cysteine thiols present by an alkylalkane thiosulfonate-type reagent, methyl methanethiol-sulfonate (MMTS), that selectively formed a disulfide bridge, thus generating an inactive thiolmethylated enzyme. When this procedure was followed, the enzyme could be undoubtedly modified at its amino by the anhydride reagent, leading to a doubly modified protein. The thiomethyl group could then be removed by reduction with dithiothreitol, yielding an enzyme modified solely on the amino residues. The amino group could be unblocked in turn by exposure to acidic pH. The different anhydrides inactivated thiolase, but only acetoacetyl coenzyme A (AcAcCoA) provided any protection against inactivation. When thiolmethylcitraconyl thiolase was reduced with dithiothreitol the enzyme remained inactive, but when the doubly modified enzyme was exposed to pH 5 then the reduction led to formation of an active enzyme. These results are interpreted as demonstrating a role for an amino group at the enzyme active site. A catalytic mechanism is proposed for the enzyme which involves the amino group.     

An Improvement in the Sensitivity of the Salkowski Reagent for Tryptamine, Tryptophan and Indoleacetic Acid

The reaction of indoles with the Salkowski reagent has been examined. It was found that the concentration of acid as well as the concentration and anionic component of the iron salt employed are critical factors in the choice of a reagent that will fail to react—or will react maximally with a given indole. Tryptamine can be reproducibly assayed with a reagent containing 0.01 M Fe(NO₃)₃ in 7.0 M HCIO₄. Two ml of this reagent are added to two ml of the sample. The absorbancy is read at 450 nm after 90 minutes under uniform light conditions. Versions of this reagent can also be used for the quantitative colorimetric determination of tryptophan or indoleacetic acid.     

Yeast enolase carboxyl modification using Woodward's reagent K

Yeast enolase is inactivated by Woodward's reagent K. Substantial protection is afforded by binding of 1 mol of "conformational" metal ion/subunit. Inactivation is correlated with modification of 13 carboxyl groups/subunit in the absence of conformational metal ion and 17 in its presence. Ten tryptic peptides labeled by Woodward's reagent K can be isolated, mostly from the C-terminal half of the protein. The changes in reactivity of these peptides produced by conformational metal ion suggest direct coordination to Glu-181 together with a contraction of the protein.     

Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent.

N-Bromoacetylethanolamine phosphate rapidly and irreversibly inactivates rabbit muscle phosphoglycerate mutase. At high molar ratios of reagent to enzyme, loss of activity (both mutase and phosphatase) approximates pseudo-first order kinetics. A rate-saturation effect is observed with half-maximal rate of inactivation occurring at 0.32 mM reagent, a value close to the Km for 3-phosphoglyceric acid. This datum and the dissociation constant of the 2,3-bisphosphoglycerate-enzyme complex, as determined from inactivation kinetics in the presence of the bisphosphate, suggest that the reagent reacts at the substrate binding site. Inactivation results from the covalent incorporation of about 0.8 mol of reagent/mol of catalytic subunit as determined with 14C-labeled reagent. Incorporation is negligible in the presence of substrate and is reduced 8-fold in the presence of 6 M urea. From amino acid analyses on acid hydrolysates of the inactivated enzyme, we have identified a sulfhydryl group as the site of alkylation. A peptide containing the essential sulfhydryl group has been isolated from a tryptic digest of the enzyme inactivated with labeled reagent; its amino acid composition is Trp1, Lys1,-Cys(Cm)1, Asp1, Ser1, Glu2, Gly1, Ala1, Leu1, Phe2.     

Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function.

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5'-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.     

SOME EFFECTS OF IODINE AND OTHER REAGENTS ON THE STRUCTURE AND ACTIVITY OF TOBACCO MOSAIC VIRUS

1. Denatured tobacco mosaic virus has a number of SH groups corresponding to its total sulfur content of 0.2 per cent. The SH groups were estimated by titration with ferricyanide, tetrathionate, and p-chloromercuribenzoate in guanidine hydrochloride solution and by reduction of the uric acid reagent in urea solution. 2. The SH groups of tobacco mosaic virus or their precursors can be abolished by reaction of the native form of the virus with iodine. 3. Tobacco mosaic virus whose SH groups have been oxidized beyond the S-S stage by iodine but whose tyrosine groups have not been converted into di-iodotyrosine groups still retains its normal biological activity as shown by the number of lesions it causes on Nicotiana glutinosa plants and by the characteristic disease produced in Turkish tobacco plants. 4. The inoculation of Turkish tobacco plants with active virus whose SH groups have been abolished by iodine results in the production of virus with the normal number of SH groups. 5. If enough iodine is added to tobacco mosaic virus or if the iodine reaction is carried out at a sufficiently high temperature, then the tyrosine groups are converted into di-iodotyrosine groups and the virus is inactivated. 6. Tobacco mosaic virus can be almost completely inactivated by iodoacetamide under conditions under which iodoacetamide reacts with few if any of the protein''s SH groups. 7. Tobacco mosaic virus is not inactivated by dilute p-chloromercuribenzoate.     

Effect of modifications of a series of amino acid radicals on the enzymatic activity of glucoamylase from Aspergillus awamori

The effect of chemical modification of various amino acid residues on the enzymatic activity of glucoamylase from Asp. awamori was studied. Modification of the carboxyl groups by taurine in the presence of water-soluble carbodiimide results in complete inactivation of the enzyme. The inactivation process includes two steps, namely non-specific modification and modification of the active center carboxyls. The rate constants of inactivation at both steps were measured in the presence and absence of the substrate, i. e. maltose. It was shown that the enzyme is inactivated by N-bromosuccinimide. Based on the data on the protection of the enzyme active center by the substrates (maltooligosaccharides of various lengths), it was concluded that the essential tryptophane residue(s) is localized in the fourth subsite. Ethoxycarbonylation, nitration and acetylation of glucoamylase do not change the catalytic activity of the enzyme. The protein was shown to contain no SH-groups.     

Reactivity of D-amino acid oxidase with 1,2-cyclohexanedione: evidence for one arginine in the substrate-binding site

D-Amino acid oxidase is inactivated by reaction with 1,2-cyclohexanedione in borate buffer at pH 8.8. The reaction follows pseudo-first-order kinetics. The present of benzoate, a substrate-competitive inhibitor of the enzyme, protects substantially against inactivation. Partial reactivation could be obtained by removal of borate and its substitution with phosphate buffer. The reaction of 1,2-cyclohexanedione with the enzyme at different inhibitor concentrations appears to follow a saturation kinetics, indicating the formation of an intermediate complex between enzyme and inhibitor prior to the inactivation process. The partially inactivated enzyme shows the same apparent Km but a decreased V as compared to the native D-amino acid oxidase. Similarly, the inhibited enzyme fails to bind benzoate. Amino acid analysis of the 1,2-cyclohexanedione-treated enzyme at various times of inactivation shows no loss of amino acid residues except for arginines. Analysis of the reaction data by statistical methods indicates that three arginine residues react with the inhibitor at slightly different rates, and that one of them is essential for catalytic activity. The presence of benzoate, while it prevents the loss of activity, reduces by one the number of arginine residues hit by the reagent in the reaction of 1,2-cyclohexanedione with D-amino acid oxidase.     

Control of autotrophic carbon assimilation in Alcaligenes eutrophus by inactivation and reactivation of phosphoribulokinase

Phosphoribulokinase in Alcaligenes eutrophus was partially inactivated when an autotrophic culture was shifted to heterotrophic growth with pyruvate as the sole source of carbon and energy. A similar response was observed on addition of various organic substrates to autotrophic cultures during the transition to mixotrophic growth. The extent of inactivation depended on the added substrate. Pyruvate or lactate caused the strongest inactivation among the tested substrates. Up to 75% of the phosphoribulokinase activity found in the autotrophic cells was lost within 30 min after supplementation of the cultures with either of these two substrates. This loss of enzyme activity was not the result of degradation of enzyme protein. Inactivation of phosphoribulokinase was accompanied by a decrease in the CO2 fixation rate of the cells. Reactivation of the enzyme occurred after exhaustion of pyruvate from the medium. Neither inactivation nor reactivation required de novo protein synthesis; however, continued energy conversion was necessary for the inactivation to occur. We suggest that the pyruvate metabolism of A. eutrophus is involved in these regulatory processes which act on phosphoribulokinase. They appear to contribute to the control of autotrophic CO2 assimilation in this organism.     

Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides

Reaction of radicals in the presence of O2, and singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, and that this can result in inactivation of thiol-dependent enzymes. The major route for the cellular removal of damaged proteins is via catabolism mediated by proteosomal and lysosomal pathways; cysteine proteases (cathepsins) play a key role in the latter system. We hypothesized that inactivation of cysteine proteases by hydroperoxide-containing oxidised proteins may contribute to the accumulation of modified proteins within cells. We show here that thiol-dependent cathepsins, either isolated or in cell lysates, are rapidly and efficiently inactivated by amino acid, peptide, and protein hydroperoxides in a time- and concentration-dependent manner; this occurs with similar efficacy to equimolar H2O2. Inactivation involves reaction of the hydroperoxide with Cys residues as evidenced by thiol loss and formation of sulfenic acid intermediates. Structurally related, non-thiol-dependent cathepsins are less readily inactivated by these hydroperoxides. This inhibition, by oxidized proteins, of the system designed to remove modified proteins, may contribute to the accumulation of damaged proteins in cells subject to oxidative stress.     

Evidence for the participation of histidine residues located in the 56 kDa C-terminal polypeptide domain of ADP-ribosyl transferase in its catalytic activity

Purified ADPRT protein was inactivated by the histidine specific reagent diethylpyrocarbonate, binding to two histidine residues, or by a relatively histidine selective photoinactivation method. Inactivation with up to 1.3 mM diethylpyrocarbonate was reversible by hydroxylamine. Enzymatic inactivation coincided with the loss of binding capacity of the enzyme protein to benzamide affinity matrix but not to DNA cellulose. Labelled diethylpyrocarbonate was identified exclusively in the 56 kDa carboxyl-terminal polypeptide where 2 out of 13 histidine residues were modified by this reagent. It is proposed that histidine residues in the 56 kDa polypeptide may participate as initiator sites for polyADP-ribosylation.     

The formaldehyde-fluorescamine method

Summary Formaldehyde reacts with primary amino groups to derivatives which are unable to react with the fluorogenic primary amino group probe, fluorescamine. Paradoxically, however, certain specific cell systems continue to display strong fluorescamine-induced fluorescence after formaldehyde pretreatment. Among such formaldehyde-fluorescamine (FF) positive cell systems are certain peptide- and protein-secreting cells as well as all hitherto investigated types of cancer cells. We have now optimized the cytochemical FF method by using microfluorometry in combination with systematically varied reaction conditions. In addition, the quantitative data indicate that in FF positive cells, formaldehyde pretreatment causes a paradoxical increase in the fluorescence yield with fluorescamine. This has tentatively been ascribed to quenching phenomena, associated with closely spaced primary amino groups. Work with alternative fluorogenic amino group probes (MDPF and OPT) show that these display the same spectrum of tissue selectivity as fluorescamine, but that the latter remains the reagent of choice for the cytochemical FF reaction.     

Involvement of arginine residues in catalysis by rat brain hexokinase

Inactivation of rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) by the arginine-specific reagent, phenylglyoxal, has been studied. Inactivation did not follow pseudo-first-order kinetics, suggesting the involvement of two or more arginine residues in catalytic function. Using [¹⁴C]phenylglyoxal, it was found that 5 of the 55 arginines per molecule of hexokinase react with this reagent, with an accompanying loss of over 90% of the catalytic activity. Virtually all of the activity loss occurs during derivatization of four relatively slower reacting arginines, with essentially no activity loss during derivatization of one rapidly reacting arginine. Inactivation by phenylglyoxal was not due to reaction with critical sulfhydryl groups in brain hexokinase since reactivity of the enzyme with the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoic acid) was not affected by prior treatment with phenylglyoxal. Comparison of amino acid composition, before and after reaction with phenylglyoxal, indicated that only the arginine content had been affected by phenylglyoxal treatment. The decrease in arginine content, measured by amino acid analysis, and the incorporation of phenylglyoxal, measured with [¹⁴C]phenylglyoxal, was consistent with the phenylglyoxal:arginine stoichiometry of 2:1 originally reported by K. Takahashi (1968, J. Biol. Chem.243, 6171–6179). Several ligands were tested and found to provide varying degrees of protection of hexokinase activity against phenylglyoxal. ATP and ADP alone provided only slight protection, but were highly effective in the presence of N-acetylglucosamine which itself gave only moderate protection. Glucose 6-phosphate and 1,5-anhydroglucitol 6-phosphate, both good inhibitors of brain hexokinase, were very effective while poorly inhibitory hexose 6-phosphates were not. Glucose was very effective, with protection afforded by other hexoses being correlated with their ability to serve as substrates (i.e., poor substrates also provided little protection against phenylglyoxal). The effectiveness of hexose 6-phosphates and hexoses in protecting the enzyme against inactivation by phenylglyoxal was related to their ability to induce conformational change in the enzyme. None of the ligands tested appreciably affected the reactivity of the rapidly reacting arginine residue. There was no correlation between the inhibition observed in the presence of various ligands and the number of arginines reacted with phenylglyoxal. The results were interpreted as indicating the involvement of two to four arginine residues in the catalytic function of brain hexokinase, possibly in the binding of anionic ligands such as ATP, ADP, or glucose 6-phosphate.     

Substrate-dependent inactivation of transaldolase activity with tetranitromethane

Transaldolase (Type III) from Candida utilis was found to be inactivated by tetranitromethane only in the presence of the substrates fructose 6-phosphate and sedoheptulose 7-phosphate. This reaction was prevented by the addition of erythrose 4-phosphate or glyceraldehyde 3-phosphate, which are known to accept dihydroxyacetone from the transaldolase-dihydroxyacetone complex, releasing free transaldolase. These results strongly suggest that tetranitromethane does not react with free transaldolase but only with the Schiff-base intermediate. After 1 min of incubation with the reagent at pH 6.0, 4 moles of nitroformate were produced per mole of inactivated enzyme. The modification, probably a nitration or an oxidation of certain amino acid residues of the complex by tetranitromethane, caused a dissociation of the dihydroxyacetone moiety from the complex without any recovery of the enzymatic activity. The fact that the reaction with tetranitromethane takes place only in the presence of substrates indicates that a substrate-mediated change of conformation occurs in transaldolase. Chemical and spectrophotometric evidence is presented showing that tetranitromethane did not modify tyrosine, cysteine, and tryptophan residues in the inactivated enzyme. From amino acid analyses it appears that histidine, serine, proline, methionine, tyrosine, and phenylalanine residues were not altered by this reagent. The possible mechanisms of modification of the transaldolasedihydroxyacetone complex and the chemical nature of the modification by tetranitromethane are discussed.     

Identification of bound pyruvate essential for the activity of phosphatidylserine decarboxylase of Escherichia coli.

Phosphatidylserine decarboxylase, an intrinsic membrane protein of Escherichia coli, catalyzes the decarboxylation of phosphatidylserine, the final step in the biosynthesis of phosphatidylethanolamine, the principal membrane lipid of this organism. The purified enzyme lacks the absorption spectrum characteristic of pyridoxal-containing enzymes, and it has now been found to contain bound pyruvate, the carbonyl function of which is essential for catalytic activity. The decarboxylase is inactivated by treatment with a number of reagents that attack carbonyl groups, including sodium borohydride. Reduction with tritiated borohydride leads to the introduction of stably bound radioactivity, which, after acid hydrolysis, has been identified as tritiated lactate by several chromatographic procedures and by treatment with lactate dehydrogenase. The enzyme resists inactivation by cyanoborohydride in the absence of substrate, but is readily inactivated by this reagent in the presence of phosphatidylserine. Under the conditions of treatment of neutral pH, cyanoborohydride does not react with carbonyl residues at an appreciable rate, but reduces imino groups much more rapidly. This finding, together with demonstrated dependence of the enzyme upon the carbonyl residue of pyruvate for activity, strongly suggests that a Schiff base is formed by addition of the amino group of phosphatidylserine to the pyruvate residue of the enzyme as an essential step in the action of the decarboxylase.     

Proteolysis upon dehydration of yeasts saccharomyces cerevisiae

The effect of dehydration on proteolysis and activity of proteases A, B and C in the cells of baker's yeast Saccharomyces cerevisiae was investigated. It can be concluded, that under investigated conditions of yeast Saccharomyces cerevisiae drying a decrease of proteases activity takes place. In cells a limited proteolysis takes place which is indicated by an increase in amino nitrogen content and a decrease of tryptophane synthase activity. Adding the protease inhibitor to yeast suspension prevents decrease of tryptophane synthase activity upon dehydration.