Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple oxidation products of sulfhydryl groups near the active site of thiolase I from porcine heart

The inactivation of porcine heart thiolase I with the disulfide reagents 5,5'-dithiobis(2-nitrobenzoate) (DTNB) and 2,2- and 4,4-dithiopyridine in 0.2 M phosphate buffer, pH 7.5, follows second-order kinetics with rate constants of 2.2 X 10(2), 25 X 10(2), and 5.8 X 10(2) M-1 min-1, respectively. Stoichiometric concentrations of the thiol-oxidizing reagent diethyl azodicarboxylate inactivate thiolase in less than 1 min at pH 7.5. The presence of saturating concentrations of the substrate acetoacetyl coenzyme A or the formation of the acetyl enzyme (a normal catalytic intermediate) results in a significant protection against the inactivation of thiolase by DTNB, 2,2-dithiopyridine, and diethyl azodicarboxylate. All five sulfhydryl residues of native thiolase react with either of the dipyridyl disulfides, but only the equivalent of 3.2 residues react with DTNB even at high concentrations and prolonged incubation times. The reaction of thiolase with DTNB leads to the formation of 1.0-1.4 mol of intrachain disulfide and 0.65 mol of mixed disulfides. After inactivation of thiolase with an equimolar concentration of diethyl azodicarboxylate, 1.2 mol of intrachain disulfide per subunit is found. No cross-linking between the subunits occurs as a result of the reaction of thiolase with DTNB or diethyl azodicarboxylate. The DTNB-inactivated enzyme can be reactivated with excess dithiothreitol while the diethyl azodicarboxylate inactivated enzyme is totally resistant to reactivation by dithiothreitol. There appear to be at least two different ways of forming inactive, oxidized enzyme products depending on the oxidant used, suggesting the possibility of multiple sulfhydryl groups at or near the active site.     

Rabbit muscle phosphofructokinase. 2. Inactivation by the affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

The reaction of the fluorescent affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit skeletal muscle phosphofructokinase results in an inactivation of the enzyme and in the covalent incorporation of up to one label/monomer. The substrates, MgATP and fructose 6-phosphate, each protect against inactivation of the enzyme, but neither diminishes the extent of covalent incorporation of the label, indicating that the inactivation is not the result of covalent incorporation of the label. Dithiothreitol reactivates the inactivated enzyme but does not reduce the extent of incorporation of the label. A determination of the number of free sulfhydryl groups on the enzyme as a function of the extent of inactivation by the reagent suggests that the inactivation is associated with the loss of two free sulfhydryl groups per phosphofructokinase monomer. The inactivation reaction appears to involve the reversible formation of an enzyme-reagent complex (Kd = 1.11 mM) prior to the conversion of the complex to inactive enzyme (k1 = 0.98 min-1). In view of the protection afforded by either substrate and the evidence suggesting the formation of an enzyme-reagent complex prior to inactivation, it would appear that the inactivation results from a reagent-mediated formation of a disulfide bond between two cysteinyl residues in close proximity, possibly in or near the catalytic site of the enzyme. The site of covalent attachment of the label appears to be the binding site specific for the activating adenine nucleotides cAMP, AMP, and ADP. The extent of covalent incorporation of the label at this site is diminished in the presence of cAMP, and phosphofructokinase modified at this site by this affinity label is no longer subject to activation by cAMP.     

Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by oxidation of a specific sulfhydryl group with potassium permanganate.

Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains four cysteine residues per subunit. Three of these react readily with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), forming an active derivative with kinetic and physical properties similar to the native enzyme, but one reacts only under denaturing conditions. Stoichiometric amounts of KMnO4 inactivate the DTNB-treated enzyme. The loss of activity is correlated with the oxidation of the remaining cysteinyl group to cysteic acid by KMnO4. Amino acid analysis indicates that no other residues are altered. The rate of inactivation of the enzyme is decreased 30-fold by saturatin g concentrations of the substrate ATP. Inorganic phosphate also protects substantially against KMnO4. Titration of the native enzyme with limiting amounts of KMnO4 shows that the sulfhydryl group essential for activity competes effectively with the other sulfhydryl groups for KMnO4. These results suggest that the essential sulfhydryl group is near the active site, and that KMnO4, a phosphate analogue, can act as an active site-directed reagent at the ATP binding site of the enzyme. The KMnO4-oxidized enzyme is more highly aggregated than untreated enzyme and fails to bind ATP appreciably.     

Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver

An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.     

Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase

Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 M-1 S-1 at 2 degrees C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with 14C-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with 14C-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethyl-maleimide. The reactive sulfhydryl groups are likely those generated by reduction of a disulfide during activation.     

The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine.

Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased. (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.     

Reactive sulfhydryl groups in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is inactivated by several thiol- and vicinal dithiol-specific reagents. Titration experiments of the enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) show the presence of reactive monothiol and vicinal dithiol groups, whose modifications lead to enzyme inactivation. The enzyme is also inactivated by N-(1-pyrenyl)iodoacetamide (PyrIAM), with a binding stoichiometry of approx. 2 mol per mol of enzyme subunit. A high level of pyrene excimer fluorescence is detected on the labeled enzyme, thus implying the reaction of the reagent with two spatially close sulfhydryl groups in the protein. The carboxykinase is not completely inactivated by different vicinal dithiol-specific reagents, thus implying a catalytically non-essential character for these groups. From substrate protection experiments of the enzyme inactivation by DTNB, PyrIAM and vicinal dithiol-specific reagents, it is concluded that the loss of enzyme activity is caused by the modification of both thiol and vicinal dithiol groups in the substrate binding region.     

The reaction of choline acetyltransferase with sulfhydryl reagents. Methoxycarbonyl-CoA disulfide as an active site-directed reagent.

The reaction of choline acetyltransferase with methoxycarbonyl alkyl disulfides leads to a progressive loss in enzyme activity as the size of the alkyl group increases from methyl to n-butyl. Reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or methoxycarbonyl coenzyme A (CoA) disulfide, leads to a total loss of enzyme activity. DTNB inactivation is biphasic (k1 = approximately 9 x 10(2) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1) with the slow phase being diminished by acetyl-CoA. Methoxycarbonyl-CoA disulfide inactivation is also biphasic (k1 = approximately 2.1 x 10(3) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1), with the rapid phase being diminished in the presence of acetyl-CoA. Inactivation by methoxycarbonyl methyl disulfide, ethyl disulfide, or hydroxyethyl disulfide, or by methyl methanethiosulfonate is not biphasic. Pretreatment of the enzyme with methyl methanethiosulfonate, which leads to a 25% loss in enzyme activity, abolishes the fast phase of DTNB inactivation, the slow phase of methoxycarbonyl-CoA disulfide inactivation, and any further inactivation by methoxycarbonyl ethyl disulfide. These results are interpreted to suggest that choline acetyltransferase contains two classes of reactive sulfhydryl groups, neither of which are required for enzyme activity.     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification

The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.     

Disulfide of thionosine triphosphate, an ATP-analog inactivating (Na+ + K+)-ATPase.

(Na+ + K+)-activated ATPase in beef brain microsomes is inactivated by the disulfide of thionosine tri[gamma-32P]phosphate, an ATP analog. The inactivation of the enzyme, which is accompanied by an incorporation of radioactivity into the membrane protein, is abolished by ATP or dithiothreitol. Since dithiothreitol restores the activity of (Na+ + K+)-ATPase, which had previously been inactivated by this ATP analog, it is concluded that thionosine triphosphate disulfide reacts with a sulfhydryl group in the ATP binding site of (Na+ + K+)-activated ATPase.     

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Identification of essential sulfhydryl residues in the primary sequence of the enzyme

The kinase and sugar phosphate exchange reactions of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by treatment with 5'-p-fluorosulfonylbenzoyladenosine or 8-azido-ATP, but activity could be restored by the addition of dithiothreitol. This inactivation was accompanied by incorporation of 5'-p-sulfonylbenzoyl[8-14C]adenosine into the enzyme that was not released by the addition of dithiothreitol. The lack of effect of ATP analogs on the ATP/ADP exchange or on bisphosphatase activity and reversal of their effects on the kinase and sugar phosphate reactions by dithiothreitol suggest that 1) they reacted with sulfhydryl groups important for sugar phosphate binding in the kinase reaction, and 2) the inactivation of the kinase by these analogs involves a specific reaction that is not related to their general mechanism of attacking nucleotide-binding sites. In addition, alkylation of the enzymes' sulfhydryls with iodoacetamide prevented inactivation by 5'-p-fluorosulfonylbenzoyladenosine, suggesting that the same thiols were involved. o-Iodosobenzoate inactivated the kinase and sugar phosphate exchange; the inactivation was reversed by dithiothreitol; but there was no effect on the bisphosphatase or nucleotide exchange, indicating that oxidation occurred at the same sulfhydryl that are associated with sugar phosphate binding. ATP or ADP, but not fructose 6-phosphate, protected these groups from modification by 5'-p-fluorosulfonylbenzoyladenosine, 8-azido-ATP, and o-iodosobenzoate. ATP also induced dramatic changes in the circular dichroism spectrum of the enzyme, suggesting that adenine nucleotide protection of thiol groups resulted from changes in enzyme secondary structure. Analysis of cyanogen bromide fragments of 14C-carboxamidomethylated enzyme showed that all radioactivity was associated with cysteinyl residues in a single cyanogen bromide fragment. Three of these cysteinyl residues are clustered in a 38-residue region, which probably plays a role in maintaining the conformation of the kinase sugar phosphate-binding site.     

Synthesis of a reactive ATP analog and its preliminary application as an affinity labeling reagent

A reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP.PCP, was synthesized in an attempt to covalently label the binding sites for adenine nucleotides, especially ATP, of various enzymes which utilize adenine nucleotides as substrates, cofactors, inhibitors or allosteric effectors. This reagent rapidly inactivated rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD), myokinase (MK), and creatine kinase (CK) under very mild conditions. Adenine nucleotide substrates prevented the inactivation. In the case of GPD, complete inactivation was observed when 1 mol of the reagent per mol of enzyme subunit was incorporated into the enzyme. These results indicate that the present ATP analog may be useful as an affinity labeling reagent for various adenine nucleotide-dependent enzymes.     

Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups

Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.     

Gamma-glutamylcysteine synthetase. Interactions of an essential sulfhydryl group

gamma-Glutamylcysteine synthetase (isolated from rat kidney) has one sulfhydryl group that reacts with 5,5'-dithiobis-(2-nitrobenzoate). This single exposed sulfhydryl group is not required for enzyme activity. The enzyme is potently inactivated by cystamine, which apparently interacts with a sulfhydryl group at the active site to form a mixed disulfide. 5,5'-Dithiobis-(2-nitrobenzoate) does not interact with the sulfhydryl group that reacts with cystamine. After the enzyme was 90% inactivated by reaction with cystamine, 3.4 mol of 5,5'-dithiobis-(2-nitrobenzoate) reacted per mol of enzyme, indicating that binding of cystamine exposes sulfhydryl groups which are apparently buried or unreactive in the native enzyme. L-Glutamate (but not D-glutamate or L-alpha-aminobutyrate) protected against inactivation by cystamine. In contrast, ATP enhanced the rate of inactivation by cystamine, and the apparent Km value for this effect is similar to that for ATP in the catalytic reaction. Studies on the structural features of cystamine that facilitate its interaction with the enzyme showed that selenocystamine, monodansylcystamine, and N-[2[2-aminoethyl)-dithio)ethyl]-4-azido-2-nitrobenzeneamine are also good inhibitors. Whereas S-(S-methyl)cysteamine-Sepharose does not interact with the enzyme (Seelig, G. F., and Meister, A. (1982) J. Biol. Chem. 257, 5092-5096), S-(S-methyl)cysteamine is a potent inhibitor; 1 mol of this compound completely inactivated 1 mol of enzyme. In the course of this work, a useful modification of the method for isolating this enzyme from kidney was developed.     

The activation of glucose dehydrogenase by p-chloromercuribenzoate

Summary P-Chloromercuribenzoate alters various reactions of rat liver glucose (hexose phosphate) dehydrogenase differently. The reagent has little effect on the glucose: NAD or the glucose: NADP oxidoreductases, doubles the rates of oxidations of galactose-6-phosphate and glucose-6-phosphate by NADP and greatly stimulates the oxidations of glucose-6-phosphate and galactose-6-phosphate by NAD. The reagent appears to react with a sulfhydryl group of the enzyme since activation is reversed and prevented by mercaptoethanol. The direct reaction of the reagent with the enzyme is indicated by its lower thermal stability in the presence of the p-chloromercuribenzoate. The size of the enzyme appears to be the same when determined by sucrose gradient centrifugation in the presence or absence of p-chloromercuribenzoate. In microsomes, the oxidation of NADH or NADPH hampers measurements of glucose dehydrogenase. Since p-chloromercuribenzoate inhibits microsomal oxidation of reduced nicontinamide nucleotides, it is possible to assay for glucose dehydrogenase accurately in the presence of the mercurial in microsomes and microsomal extracts and thus measure the effectiveness of a detergent in extracting the enzyme from microsomes.Abbreviation pcMB p-chloromercuribenzoic acid     

Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis

Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.     

Functional cysteinyl residues in human placental aldose reductase

Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 M urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species.     

Specific mixed disulfide formation with purified bovine cardiac glycogen synthase I and glutathione

Bovine cardiac glycogen-free glycogen synthase I reacts with oxidized glutathione at low temperature to partially inactivate the enzyme. Evidence is presented that a mixed disulfide between glutathione and the enzyme is formed in this reaction. A short incubation of the GSSG-treated enzyme with dithiothreitol restores full enzyme activity. The reaction with GSSG is pH dependent and the product is quite stable at neutral pH. Oxidation of one sulfhydryl group in glycogen synthase is associated with a loss of 60-70% of the enzyme activity. Further modification of protein sulfhydryls has less effect on the enzyme activity. Other low molecular weight disulfides also inactivate glycogen synthase and treatment with [35S]cystine to produce a 40% loss of enzyme activity gave rise to a single major radioactive peptide after cyanogen bromide digestion. Thus the GSSG-mediated inactivation of glycogen synthase apparently occurs through a single reactive sulfhydryl group that forms a mixed disulfide with low molecular weight disulfide molecules. Uridine 5'-diphosphate glucose and glycogen prevent the inactivation of glycogen-free glycogen synthase with GSSG, and glucose 6-phosphate retards the rate of inactivation. Reduction and reactivation of the GSSG-oxidized glycogen synthase is not affected by glycogen and it occurs readily at neutral pH with dithiothreitol, mercaptoethanol, or cysteamine. Oxidation of the reactive sulfhydryl group with GSSG has no effect on the rate of glycogen synthase phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.