Structure-activity relationships in tryptophanyl transfer ribonucleic acid synthetase from beef pancreas. Influence of the alkylation of the sulfhydryl groups on the dimer-monomer equilibrium.

Upon reaction with N-ethylmaleimide, tryptophanyl-tRNA synthetase from beef pancreas dissociates into subunits. At pH7, the rate of the dissociation is close to both the reaction rate of the buried--SH groups and the rate of inactivation (Iborra, F., Mourgeon, G., Labouesse B., and Labouesse, J. (1973) Eur. J. Biochem. 39, 547-556). The pH and enzyme concnetration dependences of the reaction rate of the 16 cysteinyl residues of the enzyme as well as that of its inactivation support the idea that inactivation by alkylation of the--SH groups is due essentially to the dissociation of the protein into inactive subunits and not to the chemical blocking of a catalytic residue. This is confirmed by the independence on N-ethylmaleimide concentration of the reaction of the buried--SH groups and of the inactivation of the enzyme at high N-ethylmaleimide concentration. The dissociation becomes in this case the rate-limiting step of the chemical reaction. The monomeric structure is stabilized by the blocking of the--SH groups exposed during the dissociation. The dissociation constant of the dimeric enzyme is progressively increased during the alkylation. The tightness of the associated structure depends on the protonation of groups titrating between pH 7 and pH 9.     

The role of thiol groups in the structure and mechanism of action of arginine kinase

1. A detailed study of the reaction of iodoacetamide with arginine kinase has been carried out. 2. The enzyme contains five reactive thiol groups per 37000g. of protein, all of which can be alkylated. 3. Below pH8.5 loss of activity is substantially independent of pH and can be correlated with the alkylation of a single pH-independent thiol. 4. One catalytic site per enzyme molecule is inferred. 5. The progress curves of the alkylation reaction are polyphasic and reveal a pH-and time-dependent sequential release of thiols which is dependent upon the alkylation of the first pH-independent thiol. This is supported by electrophoretic investigations. 6. Comparison of alkylation rate and rate of loss of activity suggests that two thiol groups are not essential for catalytic activity. Variability in enzyme preparations with respect to alkylation rate appears to be associated with these two groups. 7. A complex protection pattern is revealed by the effects of various substrate combinations on rates of alkylation and of loss of activity. It is inferred that two thiol groups participate in conformational changes and nucleotide interactions. 8. Comparison with creatine kinase suggests a fundamentally similar catalytic mechanism, although for arginine kinase certain additional restrictions are necessary because of the protection observed with nucleotide substrates.     

Identification of the essential cysteine residue in Klebsiella aerogenes urease.

During reaction with [14C]iodoacetamide at pH 6.3, radioactivity was incorporated primarily into a single Klebsiella aerogenes urease peptide concomitant with activity loss. This peptide was protected from modification at pH 6.3 by inclusion of phosphate, a competitive inhibitor of urease, which also protected the enzyme from inactivation. At pH 8.5, several peptides were alkylated; however, modification of one peptide, identical to that modified at pH 6.3, paralleled activity loss. The N-terminal amino acid sequence and composition of the peptide containing the essential thiol was determined. Previous enzyme inactivation studies of K. aerogenes urease could not distinguish whether one or two essential thiols were present per active site (Todd, M. J., and Hausinger, R. P. (1991) J. Biol. Chem. 266, 10260-10267); we conclude that there is a single essential thiol present and identify this residue as Cys319 in the large subunit of the heteropolymeric enzyme.     

Aminoethylation in model peptides reveals conditions for maximizing thiol specificity

Control of pH in aminoethylation reactions is critical for maintaining high selectivity towards cysteine modification. Measurement of aminoethylation rate constants by liquid chromatography mass spectrometry demonstrates reaction selectivity of cysteine>amino-terminus>histidine. Lysine and methionine were not reactive at the conditions used. For thiol modification, the acid/base property of the gamma-thialysine residue measured by NMR results in a 1.15 decrease in pK(a) (relative to a lysine residue). NMR confirms ethylene imine is the reactive intermediate for alkylation of peptide nucleophiles with bromoethylamine. Conversion of bromoethylamine into ethylene imine prior to exposure to the target thiol, provides a reagent that promotes selectivity by allowing precise control of reaction pH. Reaction selectivity plots of relative aminoethylation rates for cysteine, histidine, and N-terminus imine demonstrate increasing alkaline conditions favors thiol modification. When applied to protein modification, the conversion of bromoethylamine into ethylene imine and buffering at alkaline pH will allow optimal cysteine residue aminoethylation.     

Irreversible inhibition of fatty acid synthase from rat mammary gland with S-(4-bromo-2,3-dioxobutyl)-CoA. Effect on the partial reactions, protection by substrates and stoichiometry studies.

Fatty acid synthase from lactating rat mammary gland is rapidly and irreversibly inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. Of the seven partial reactions catalysed by the enzyme, the inhibition of the overall catalytic activity is closely paralleled only by inhibition of the beta-oxoacyl synthase (condensing) partial reaction. Three partial reactions. Beta-oxoacyl reductase, beta-hydroxyacyl dehydratase and enoyl reductase, are inhibited to a modest degree. The three partial reactions known to involve an acyl-CoA/CoA-binding site, acetyl acyltransferase, malonyl acyltransferase and palmitoyl thioesterase, are not inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. The modification process does not cause the enzyme to dissociate into catalytically incompetent monomers. Stoichiometric studies suggest that approx. 6 mol of reagent are incorporated per mol of totally inhibited enzyme (dimer). The formation of acylated enzyme from either acetyl-CoA or malonyl-CoA protects the enzyme equally well against S-(4-bromo-2,3-dioxobutyl)-CoA. Also, pretreatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid), a thiol-specific reagent reported to block essential thiol groups in the condensing partial reaction, protects against inhibition by the reagent. On the other hand, the presence of up to 770 microM-S-acetonyl-CoA or dethio-CoA does not protect the enzyme from irreversible inhibition. Together, the results suggest that the primary inhibitory process is a bimolecular reaction resulting in alkylation of essential thiol groups in the condensing partial reaction: this process does not require the obligatory formation of a Michaelis-Menten complex of enzyme and reagent before the alkylation reaction.     

Peroxide modification of monoalkylated glutathione reductase. Stabilization of an active-site cysteine-sulfenic acid

Hydrogen peroxide reacts with two-electron reduced glutathione reductase (GR EH2 species) to give the native oxidized enzyme (E) without detectable intermediates. Prior alkylation of the EH2 interchange thiol with iodoacetamide, however, dramatically changes both the course and overall rate of the peroxide reaction. This oxidation, monitored spectrally, is characterized by an intermediate (EHRint) with enhanced long wavelength absorbance extending to 800 nm. This species decays in a second peroxide-dependent phase to an enzyme form (EHRox) easily distinguished from E. Quenching experiments with catalase allow the isolation of a stable mixture consisting of 36% monoalkylated GR (EHR), 60% EHRint, and 4% EHRox; NADPH titration and anaerobic dithiothreitol addition lead to quantitative reduction of EHRint to EHR, and there is an increase in thiol titer of 0.8-SH/FAD on NADPH reduction. Of the four titratable thiols present in EHR, 2.7 are lost on oxidation to EHRox and 0.7-0.8 mol of cysteic acid/FAD is formed. On the basis of these and other observations, we conclude that alkylation of the EH2 interchange thiol, which blocks disulfide formation, allows peroxide reaction at the remaining charge-transfer thiol to proceed via a stabilized cysteine-sulfenic acid intermediate (EHRint), which undergoes further oxidation to the corresponding cysteic acid (EHRox).     

Essential sulfhydryl groups of rat liver monoamine oxidase.

The inhibition by some thiol reagents of partly purified mitochondrial monoamine oxidase (MAO) (EC 1.4.3.4) from rat liver was studied, and the molar content of sulfhydryl groups in the enzyme determined. Sodium nitroprusside and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the enzyme, apparently reversibly, while sodium arsenite was not inhibitory. Concentrations of the respective inhibitors causing 50% inhibition after 15 min of preincubation with the enzyme at pH 7.0 and 37 degrees C are 5.80 times 10(-4) M and 4.35 times 10(-5) M. The thiol compounds cysteine, dithiothreitol, and 2-mercaptoethanol did not inhibit MAO. The average number of sulfhydryl groups per mole of enzyme, determined by reaction with DTNB, increased from 3.6 +/- 0.2 freely reacting sulfhydryl groups (n = 4) to 18.4 to total sulfhydryl groups (n = 2) on denaturation with 8 M urea.     

Identification of the amino acid residues essential for the activity and the interconversion of the molecular forms of biliverdin reductase

Biliverdin reductase (molecular form 1, EC 1.3.1.24, bilirubin:NAD(P)+ oxidoreductase) carries three thiol residues. Only one of them could be alkylated when a ratio N-ethylmaleimide (NEM)/mol enzyme's SH = 90 was used. The alkylation of this thiol group inhibited the conversion of molecular form 1 to its dimer, molecular form 3; however, it did not inhibit the enzymatic activity. At a ratio of NEM/enzyme's SH = 300, two thiol residues were alkylated and the activity of the enzyme was totally inhibited. The third thiol group could not be alkylated either by NEM or by iodoacetamide. Biliverdin as well as the co-substrate NADPH protected the thiol residue essential for the enzymatic activity from alkylation. Spectroscopic evidence was obtained that this thiol group binds covalently to the C-10 of biliverdin to form a rubinoid adduct. The presence of a lysine residue, which is also essential for the enzymatic activity, could be inferred from the fact that by reduction of the Schiff base formed by the enzyme with pyridoxal phosphate the catalytic activity was irreversibly abolished. The location of a lysine residue in the vicinity of the thiol group involved in the catalytic activity was evident when the enzyme was treated with o-phthalaldehyde. The inactivation of the enzymatic activity was coincident with the formation of the fluorescent isoindole derivative which originates when the thiol and epsilon-NH2 groups are located about 3 A apart. The presence of a positively charged ammonium ion in the vicinity of the NADPH binding site was inferred from the shifts in the UVmax of NADPH from 340 nm to 327 nm and of 3-acetyl NADPH from 360 nm to 348 nm when the pyridine nucleotides bind to the reductase. The involvement of arginine residues in the enzymatic activity was established by inhibition of the latter after reaction with butanedione. This inhibition was totally protected by NADPH but not by biliverdin. The similarity of the structural features of biliverdin reductase with those of several dehydrogenases is discussed.     

Ion-pair formation as a source of enhanced reactivity of the essential thiol group of D-glyceraldehyde-3-phosphate dehydrogenase.

The reactivity and the mode of activation of the essential--SH group (Cys-149) of D-glyceraldehyde-3-phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgár, L., FEBS Lett. 38, 187-190 (1974)], capable of detecting the dissociated form of the thiol group in proteins. Alkylations of Cys-149 of NAD-free D-glyceraldehyde-3-phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys-149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK1 equals 5.5, pK2 equals 8.2). It is concluded that there are two reactive forms of the--SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion-pair composed of a mercaptide ion and some base, possibly the imidazolium group of His-176. The ion-pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion. The two reactive forms are transformed into each other with pK2 equals 8.2. The ion-pair decomposes to a nondissociated thiol group and a protonated base with pK1 equals 5.5. In the presence of NAD, only the pH-rate profile of alkylation of D-glyceraldehyde-3-phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK1 and an upward shift in pK2 indicate that the ion-pair exists in a somewhat wider pH-range in the enzyme-coenzyme complex. An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.     

The reactivity of thiol groups and the subunit structure of aldolase

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-(14)C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5'-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.     

Affinity labeling of a previously undetected essential lysyl residue in class I fructose bisphosphate aldolase.

The affinity label N-bromoacetylethanolamine phosphate (BrAcNHEtOP) has been used previously at pH 6.5 to identify His-359 of rabbit muscle aldolase as an active site residue. We now find that the specificity of the reagent is pH-dependent. At pH 8.5, alkylation with 14C-labeled BrAcNHEtOP abolishes both fructose-1,6-P2 cleavage activity and transaldolase activity. The stoichiometry of incorporation, the kinetics of inactivation, and the protection against inactivation afforded by a competitive inhibitor or dihydroxyacetone phosphate are consistent with the involvement of an active site residue. A comparison of 14C profiles obtained from chromatography on the amino acid analyzer of acid hydrolysates of inactivated and protected samples reveals that inactivation results from the alkylation of lysyl residues. The major peptide in tryptic digests of the inactivated enzyme has been isolated. Based on its amino acid composition and the known sequence of aldolase, Lys-146 is the residue preferentially alkylated by the reagent. Aldolase modified at His-359 is still subject to alkylation of lysine; thus Lys-146 and His-359 are not mutually exclusive sites. However, aldolase modified at Lys-146 is not subject to alkylation of histidine. One explanation of these observations is that modification of Lys-146 abolishes the binding capacity of aldolase for substrates and substrate analogs (BrAcNHEtOP), whereas modification of his-359 does not. Consistent with this explanation is the ability of aldolase modified at His-359 to form a Schiff base with substrate and the inability of aldolase modified at Lys-146 to do so. Therefore, Lys-146 could be one of the cationic groups that functions in electrostatic binding of the substrate's phosphate groups.     

Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2′-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols

1. The u.v.-spectral characteristics of 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs(2)), 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py), 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5-8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py-2-SH and Py-4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs(2) is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5-8 at 25 degrees C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py-S-S-2-Py and 4-Py-S-S-4-Py in the pH range 2.5-8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine-2-Py-S-S-2-Py reaction (k(pH4)/k(pH8)=0.015) and for the papain-4-Py-S-S-4-Py reaction (k(pH4)/k(pH8)=0.06) are less than 1, that for the papain-2-Py-S-S-2-Py reaction is greater than 1 (k(pH4)/k(pH8)=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py-S-S-2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.     

Enzymic and physicochemical properties of Streptomyces griseus trypsin.

Streptomyces griseus trypsin has been isolated from Pronase by ion-exchange chromatography on CM-Sephadex and SE-Sephadex. The isolated enzyme was homogeneous by the criteria tested except for a low degree of contamination by an enzyme with nontryptic activity. The latter could be partially resolved by chromatography on Bio-Rex 70. The molar absorbancy at 280 nm was found to be 3.96 times 10-4 M-1/cm and the E1cm1% was found to be 17.3. The molecular weight was 22,800 plus or minus 800. The enzyme was found to be stable at 0 degrees from pH 2 to 10. At 30 degrees the enzyme was maximally stable at pH 3-4 and significantly stabilized in the neutral and alkaline range by 15 mM Ca2+. Some evidence was obtained for a reversible denaturation of the enzyme at pH 12.0 and 2.0. The K-m for N-alpha-benzoyl-L-arginine ethyl ester at pH 8.0 in 20 mM CaCl2-0.1 M KCl-10 mM Tris-HCl buffer at 30 degrees was found to be 7.7 plus or minus 1.9 times 10-6 M and the esterase activity was observed to be dependent on an ionizing group with pK-a equals 5.85. In 2H2O this pKa was increased to 6.35 and the rate of hydrolysis dicreased threefold. The rate of hydrolysis was independent of pH between 8 and 10. The inhibition of the enzyme with L-1-chloro-3-tosylamido-4-phenyl-2-butanone was shown to be associated with the alkylation of its single histidine residue. This residue is present in a homologous amino acid sequence as the active-site histidine in trypsin and chymotrypsin. Optical rotatory dispersion and circular dichroism measurements over the pH range 5.3-10.5 indicated no significant conformational change until the pH was increased above 10.1. The observation that, under the conditions tested, acetylation and carbamylation of the NH2-terminal valine were incomplete is consistent with the view that this group is buried as an ion pair and only becomes available for deprotonation and reaction upon denaturation of the enzyme at pH values greater than 10.0.     

Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes.

A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285 000) is made up of eight apparently identical subunits of Mr 35 000. The enzyme is sensitive to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (approximately equal to 12% of control) when assayed in buffers devoid of Zn2+. Addition of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 microM-Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constant for E X S X Pb2+ complex) = 25.3 +/- 3.0 microM; KiS (dissociation constant for E X Pb2+ complex) = 9.0 +/- 2.0 microM]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.     

A method for the estimation of thiol esters.

1. A method is described for the estimation of thiol ester groups. The thiol ester is converted into the corresponding thiol by reaction with ammonia; the thiol is then titrated amperometrically with mercuric chloride. 2. The method may be used in the presence of SH and S.S groups. The SH groups are titrated at pH3 in the presence of excess of chloride; under these conditions thiol esters do not react with mercuric chloride. Thiol ester plus thiol is then estimated by titration after reaction with ammonia. Finally, titration after reaction with ammonia and sulphite gives the thiol ester plus thiol plus disulphide. 3. The procedure has been applied to glyceraldehyde phosphate dehydrogenase. The enzyme was found to contain 15-16 SH groups/mol. and no S.S groups. After reaction with acetyl phosphate 1.8-3.5 thiol ester groups were detected, the number depending on the conditions of acetylation. In the absence of bound NAD, the number of thiol ester groups formed was 1.8/mol., although a value of 2.9 labile acetyl groups/mol. was given by the method of Lipmann & Tuttle (1945). The presence of thiol ester groups in the S-(d-3-phosphoglyceryl)-enzyme was also demonstrated.     

Synthesis of chloromethyl ketone derivatives of fatty acids. Their use as specific inhibitors of acetoacetyl-coenzyme A thiolase, cholesterol biosynthesis and fatty acid synthesis.

A general route for the synthesis of chloromethyl ketone derivatives of fatty acids is described. 5-Chloro-4-oxopentanoic acid, 7-chloro-6-oxoheptanoic acid, 9-chloro-8-oxononanoic acid and 11-chloro-10-oxoundecanoic acid were synthesized by this method and tested as covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The K1 decreased by approx. 20-fold for each pair of methylenes added to the chain length, showing that the initial stage in inhibitor binding occurs at a non-polar region of the protein. This region is probably located at the enzyme active site, since inhibition was prevented by acetoacetyl-CoA or acetyl-CoA but not by CoA. The site of modification by chloromethyl ketone derivatives of fatty acids is restricted to a thiol group, since inactivation of the enzyme was prevented by reversible thiomethylation of the active-site thiol. In contrast, an amino-directed reagent, citraconic anhydride, still inactivated the enzyme, even when the active-site thiol was protected. Evidence that the enzyme thiol was particularly reactive came from studies on the pH-dependence of the alkylation reaction and thiol-competition experiments. Inhibition of the enzyme proceeded suprisingly well at acidic pH values and a 10(5) molar excess of external thiol over active-site thiol was required to prevent inhibition by 0.3 mM-9-chloro-8-oxononanoic acid. In addition to inhibiting isolated acetoacetyl-CoA thiolase, in hepatocytes the chloromethyl ketone derivatives of fatty acids also inhibited chloresterol synthesis, which uses this enzyme as an early step in the biosynthetic pathway. In isolated cells, the chloromethyl ketone derivatives of fatty acids were considerably less specific in their inhibitory action compared with 3-acetylenic derivatives of fatty acids, which act as suicide inhibitors of acetoacetyl-CoA thiolase. However, 9-chloro-8-oxononanoic acid was also an effective inhibitor of both hepatic cholesterol and fatty acid synthesis in mice in vivo, whereas the acetylenic fatty acid derivative, dec-3-ynoic acid, was completely ineffective. The effective inhibitory dose of 9-chloro-8-oxononanoic acid (2.5-5 mg/kg) was substantially lower than the estimated LD50 for the inhibitor (100 mg/kg).     

Sequential protection-modification method for selective sulfhydryl group derivatization in proteins having more than one cysteine

The method of Smith and Hartman [J. Biol. Chem., 263, 4921-4925 (1988)] for introducing the non-natural lysine analog, S-(2-aminoethyl)cysteine, into specific sites in proteins by alkylation of a genetically introduced cysteine with 2-bromoethylamine has been generalized to be applicable to proteins containing one or more endogenous cysteines. The target cysteine residue introduced at the active site of aspartate aminotransferase is protected by bound cofactor. The enzyme is partially unfolded in low concentrations of urea, and the non-active site cysteine residues derivatized by a reversible thiol protecting reagent. The active site cysteine is then exposed and alkylated in 6 M urea. Enzyme activity is regenerated by removal of the thiol protecting groups and refolding of the protein.     

The reaction of N-ethylmaleimide at the active site of succinate dehydrogenase.

Since 1938 mammalian succinate dehydrogenase has been thought to contain thiol groups at the active site. This hypothesis was questioned recently, because irreversible inhibition by bromopyruvate and N-ethylmaleimide appeared not to satisfy the requisite criteria for reaction at the active site. These recent observations of incomplete inactivation of succinate dehydrogenase by N-ethylmaleimide and incomplete protection by substrates can, however, be explained adequately by the presence of oxalacetate and other strong competitors of the inactivation process in the enzyme used in these studies. Substrates, competitive inhibitors, and anions which activate succinate dehydrogenase protect the enzyme from inhibition by N-ethylmaleimide. Inhibition of succinate dehydrogenase by N-ethylmaleimide involves at least two second order reactions which are pH dependent, with pKa values of 8.0 to 8.2. This pH dependence, the known reactivity of N-ethylmaleimide toward thiols, and the protection by substrate and competitive inhibitors indicate that sulfhydryl residues are required for catalytic activity and perform an essential, not secondary, role in the catalysis. Just as the presence of tightly bound oxalacetate prevents inhibition by N-ethylmaleimide, alkylation of the sulfhydryl residue(s) at the active site prevents the binding of [14C]oxalacetate. Thus, these thiol groups at the active site also may be the site of tight binding of oxalacetate during the activation-deactivation cycle.     

The Cys-Xaa-His metal-binding motif: {<Emphasis Type="Italic">N</Emphasis>} versus {<Emphasis Type="Italic">S</Emphasis>} coordination and nickel-mediated formation of cysteinyl sulfinic acid

A series of peptide ligands containing the sequence -Cys-Xaa-His- (CXH; Xaa=Gly or Lys) has been prepared and the coordination chemistry of these peptides with nickel(II) investigated. Selective protection of either the N-terminal cysteine thiol or amine group gave complexes with amino or thiolato coordination, respectively, to nickel(II). Insertion of CGH into a pentapeptide, N-acetyl-Ala-Cys-Gly-His-Ala-CONH₂, allowed the formation of a square-planar thiolato Cys-Gly-His complex with nickel(II) in an internal position of the peptide. Inclusion of an N-terminal cysteine residue with a free amino terminus gave rise to pH- and dioxygen-dependent coordination behavior. Solutions of CGH-CONH₂ with nickel(II) at neutral pH yielded a red nickel-thiolate complex, but at higher pH (8.5 or above) or with exposure to dioxygen, yellow nickel complexes with N-terminal amino coordination were observed. The disulfide-bridged dimers formed from Ni(CGH-CONH₂) in the presence of air were characterized and found to have the typical coordination found in the amino-terminal binding motif of the serum albumins. Nickel(II) coordination and thiol reactivity were also studied by determination of rates of thiol alkylation and by monitoring air oxidation in the presence of various metals. Zinc(II) effectively inhibits thiol alkylation and oxidation (disulfide formation) in all the peptides studied. Nickel(II) inhibits aerobic oxidation and alkylation of N-terminal protected peptides such as N-acetyl-Cys-Gly-His, but does not inhibit air oxidation of free amino terminal peptides such as Cys-Gly-His. Instead, nickel(II) mediates the formation an additional product under aerobic conditions, a cysteinesulfinic acid.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations CGH cysteinylglycylhistidine - GGH glycylglycylhistidine - Xaa any amino acid     

Electrophilic amination of a single methionine residue located at the active site of D-amino acid oxidase by O-(2,4-dinitrophenyl)hydroxylamine

O-(2,4-Dinitrophenyl)hydroxylamine is a rapid active-site-directed inhibitor of D-amino acid oxidase: modification results in specific incorporation of an amine group into an accessible nucleophilic residue with concomitant release of 2,4-dinitrophenol. The reaction is prevented by the competitive inhibitor benzoate, indicating an active-site-directed reaction. A stoichiometry of 1-1.5 mol of amine residues per enzyme bound flavin adenine dinucleotide monomer was observed at pH 7.0. Amino acid and sequence analyses show that His-217 is not the target of the modification reaction. Dependence of the modification on pH, model studies on functional groups present on amino acids, and thiolysis studies on aminated enzyme collectively indicate that the modification is located on a methionine residue at or near the active site of the enzyme. Aminated enzyme, although spectrally similar to native enzyme, exhibits a 7-9-nm blue shift in the 455-nm flavin absorption. Benzoate perturbs the spectrum of aminated enzyme, but binding relative to native enzyme is much weaker (Kd ca. 300 times greater at pH 8.0).