Enzyme Sulfhydryl Groups Research Articles

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: Chromatographic, electrophoretic, and sedimentation behavior of active and inactive enzyme forms

This paper describes the study of a highly purified pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Short-term storage (24 h at 4 °C) of the purified enzyme in the absence of dithiothreitol, a sulfhydryl reducing agent, led to considerable losses of enzyme activity. Most of the lost activity could be regained, however, by incubating the enzyme with 50 m m dithiothreitol. Enzyme stabilization by dithiothreitol and reactivation by dithiothreitol were enhanced in the presence of phosphate buffer. Severe enzyme inhibition was produced by micromolar concentrations of sulfhydryl group reagents. Chromatographic, electrofocusing, and sucrose gradient centrifugation experiments revealed that the enzyme has a molecular weight of about 26,000, an isoelectric point of 4.7, and a sedimentation coefficient of 2.5. These experiments were also carried out with enzyme preparations which had been almost completely inactivated by means of dialysis to remove dithiothreitol. Enzyme preparations of this type displayed at least one additional enzyme form. This form(s) was inactive but capable of being partially reactivated by dithiothreitol. The inactive form(s) exhibited the same apparent molecular weight as the native enzyme but possessed a higher isoelectric point (5.7). A working hypothesis was presented which states (1) that inactive enzyme forms arise because of disulfide bond formation, (2) that enzyme sulfhydryl groups are less susceptible to oxidation in the presence of phosphate buffer, and (3) that enzyme reactivation by dithiothreitol results from the regeneration of critical enzyme sulfhydryls.

Evidence for covalently cross-linked dimers and trimers of enzyme I of the Escherichia coli phosphotransferase system

Enzyme I of the bacterial phosphotransferase system catalyzes transfer of the phosphoryl moiety from phosphoenolpyruvate to both of the heat-stable phosphoryl carrier proteins of the phosphotransferase system, HPr and FPr. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-pressure liquid chromatography, we demonstrated the existence of covalently cross-linked enzyme I dimers and trimers. Enzyme I exchange assays and phosphorylation experiments with [32P]phosphoenolpyruvate showed that covalent dimers and trimers are catalytically active. Inhibitors of the enzyme I-catalyzed phosphoenolpyruvate-pyruvate exchange block the phosphorylation of enzyme I dimers and trimers. Inhibition of the activity of enzyme I by N-ethylmaleimide, but not that by p-chloromercuriphenylsulfonate, could be overcome by high concentrations of enzyme, suggesting that N-ethylmaleimide modification changes the associative properties of enzyme I. We present evidence for two distinct classes of sulfhydryl groups in enzyme I.

Effect of sulfhydryl modification on the activities of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Alkylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with p-mercuribenzoate caused a rapid stimulation of the kinase and an inhibition of the bisphosphatase. At later times, the kinase activity also became inhibited. In contrast, treatment with N-ethylmaleimide abolished kinase activity but had no effect on the bisphosphatase. Selective modification of residues involved in the kinase reaction was also seen with iodoacetamide, which caused a 10-fold stimulation of the kinase Vmax without affecting the bisphosphatase. The stimulatory effect of carboxyamidomethylation was seen when the kinase was assayed in the presence of inorganic phosphate, an allosteric activator of the enzyme. The iodoacetamide-treated enzyme had a 10-20-fold higher Km for fructose 6-phosphate than the native enzyme and the Ki for fructose 2,6-bisphosphate was also increased. However, the adenine-nucleotide site did not seem to be affected since there was no change in the Km for ATP, the Ki for ADP, or the adenine-nucleotide exchange. There was also a direct correlation between the incorporation of [14C]acetamide into the enzyme and activation of the kinase. The residues modified by iodoacetamide were shown to be cysteines by the exclusive appearance of carboxymethylcysteine in protein hydrolysates. Activation was associated with alkylation of 2 cysteines/subunit, of the 12 which could be alkylated after denaturation/reduction. Iodoacetamide-activated kinase was inhibited by ascorbate/Fe3+, which has been shown to modify sulfhydryl groups in the native enzyme, with concomitant loss of kinase activity.

Selective inactivation of rabbit reticulocyte initiation factor eIF-3 by helenalin

Helenalin, a sesquiterpene lactone which reacts primarily with exposed sulfhydryl groups, was shown to be an effective inhibitor of protein synthesis in rabbit reticulocyte lysates. Optimal inhibition required a 30 min preincubation in the absence of any added thiol compound. β-Mercaptoethanol was more effective than reduced glutathione in protecting enzyme sulfhydryl groups from inactivation by helenalin. Using partially fractionated systems, it was possible to show that helenalin had no effect on the elongation reactions or on the formation of the ternary initiation complex. However, the conversion of the ternary complex to the 48 S initiation complex was strongly inhibited. In this assay, only the initiation factor(s) were sensitive to helenalin. Using an assay system which requires all the initiation factors for optimal activity it was possible to show that the 0–40% ammonium sulfate cut of intiation factors (containing eIF-3 and eIF-4B) was sensitive to helenalin, while the 40–50% ammonium sulfate cut (containing eIF-2 and eIF-5) was not. Both ammonium sulfate cuts were equally sensitive to inhibition by the sulfhydryl reagent N-ethylmaleimide. Three purified rabbit reticulocyte initiation factors were then tested in the same assay system. Only eIF-3 showed appreciable sensitivity to helenalin, while eIF-2, eIF-3 and eIF-4B were all sensitive to inactivation by N-ethylmaleimide. These data suggest that helenalin may possess a relatively high degree of specificity as a sulfhydryl reagent.

Improved purification and sulfhydryl analysis of thiosulfate reductase

Thiosulfate reductase purified 900-fold from an extract of baker's yeast by a new procedure consisted of three charge-isomeric species, all with catalytic activity. Amino acid analysis of thiosulfate reductase and titrations with 5,5′-dithiobis(2-nitrobenzoate) and iodo[ 14C]acetate indicated only one cysteine residue per enzyme molecule. Alkylation of this cysteine with either iodoacetate or iodoacetamide did not inactivate the enzyme, indicating that sulfur-sulfur bond cleavage in the thiosulfate substrate does not require an enzymic sulfhydryl group as attacking nucleophile.

Corrinoid catalysis of thiol oxidation

Under appropriate conditions, thiols can react with corrinoids to form relatively stable complexes [1–7], or reduced corrinoids [1, 2, 8–11]. If alkyl halides are present during the reduction of corrinoids by thiols, alkyl corrinoids are produced [4, 9, 12, 13]. In the presence of oxidizing substrates, corrinoids will efficiently catalyze the oxidation of thiols to their corresponding disulfides [14–18]. In view of the important role that enzyme sulfhydryl groups play in corrinoid–coenzyme-dependent catalysis [19, 20], we have further characterized aerobic thiol oxidation catalyzed by a selected group of biologically important corrinoids and have determined the stoichiometry of the reaction. Hydrogen peroxide and superoxide have been identified as reaction products during the aerobic catalysis of 2-mercaptoethanol [ME, 21] by the corrinoids listed in Table I. The reactions were conducted in a polarographic cell equipped with a Clark O 2 electrode [22]. The corrinoids used to initiate the reactions were prepared as described previously [23, 24] and their purity was determined by hplc [25]. H 2O 2 was detected by adding catalase to the reaction system. This resulted in an abrupt increase in O 2 concentration which is consistent with the catalytic activity of the enzyme. As anticipated, the pseudo-first order rate constant (k 1) of O 2 consumption in the presence of catalase decreased 50 per cent. Using a similar approach, superoxide dismutase was used in an attempt to detect the presence of O − 2 as a reaction product. This was unsuccessful except for one corrinoid, namely Aq-Cbl (Table I). Thus, during the catalysis of ME oxidation by Aq-Cbl, O − 2 is the primary reaction product. In the presence of dismutase, k 1 for the Aq-Cbl-catalyzed reaction decreased approximately 50 per cent which is consistent with the catalytic activity of this enzyme. H 2O 2 was also detected during Aq-Cbl-catalyzed oxidation of ME and may have been produced by the spontaneous dismutation of O − 2. Disulfide bond formation was monitored spectrophotometrically [26] during the oxidation of DTE. Pseudo-first-order rate constants for O 2 disappearance and for DTE ox appearance were in good agreement (Table II). These results, which differ from previously published studies [16, 17, 27], suggest that the reactions and their stoichiometries for aerobic oxidation of mono- and dithiols by corrinoids are: 2 RSH + O 2 → RSSR + H 2O 2 (1) ▪ t001 Pseudo-First-Order Rate Constants, pH Optima, Products and Photosensitivity for Catalysis of ME Oxidation by Corrinoids. a Group Catalyst k 1, sec −1 pH Optimum Reduced Product k 1, sec −1 (after photolysis) b I AdoCbl 0.003 Broad H 2O 2 0.102 (20 sec) MeCbl 0.003 7.0 c H 2O 2 0.184 (20 sec) II CNCbl 0.080 8.0 c H 2O 2 0.080 (60 sec) AqCbl 0.180 8.4 HO 2 0.180 (20 sec) AdoCbi 0.23 H 2O 2 177.0 (60 sec) MeCbi 0.56 11–13 H 2O 2 122.0 (90 sec) III CNCbi 191 8.5–9.5 H 2O 2 190.0 (30 sec) (Aq) 2Cbi 211 8.8 H 2O 2 210.0 (60 sec) a ME = 2.5 × 10 −2; 25 ± 0.2 °C. b At the midway point of O 2 consumption, the polarographic cell was irradiated with light from a tungsten-filament high intensity lamp for the time indicated. c Measurement was made at the indicated pH only. t002 Pseudo-First-Order Rate Constants for the Disappearance of O 2 and the Formation of Oxidized DTE. 1 Catalyst O 2 Disappearance k 1, sec −1 2 DTE ox Formation k 1, sec −1 3 Ratio O 2 DTE ox AqCbl 0.57 0.63 0.91 AdoCbi 0.49 0.46 1.07 (Aq) 2Cbi 567 597 0.95 1 Initial substrate DTE = 1.25 × 10 −2 M; pH = 8.0; 25 ± 0.2 °C. 2 The rate of O 2 disappearance was determined polarographically. 3 The rate of oxidized DTE formation was determined spectrophotometrically. The results of dismutase study and the data in Table II suggest that, in the case of AqCbl, the reactions are: ▪ Autocatalytic behavior was not observed for reactions catalyzed by the alkylcobalamins (AdoCbl and MeCbl) and the alkylcobinamides (AdoCbi and MeCbi) suggesting that the carboncobalt bond remains intact during the catalytic cycle. Similar observations have been made by Pellizer {et al.} [28] for the catalysis of cysteine oxidation by the model compound [MeCo(tn)H 2O] +. The corrinoids in Table I have been grouped according to their catalytic activity. The alkylcobalamins (AdoCbl and MeCbl; Group I) are poor catalysts. The second group of corrinoids includes CNCbl, AqCbl, AdoCbi and MeCbi. The most active catalysts (CNCbi and (Aq) 2Cbi; Group III) are cobinamides which lack covalently attached ligands. Thus, the catalytic activity of corrinoids with respect to thiol oxidation appears to be determined by the number (1 vs. 2) and accessibility (coordinate vs. covalent) of axial-ligand positions.

Inhibition by ozone of the acylation of glycerol 3-phosphate in mitochondria and microsomes from rat lung

The synthesis of lipids from [U- 14C]glycerol 3-phosphate by mitochondrial or microsomal fractions from rat lung was inhibited by ozone. The susceptible reaction was the first acylation of glycerol 3-phosphate. Enzymes unaffected by the ozone exposure included: acyl-CoA thioesterase, acyl-CoA thiokinase, acyl-CoA:acylglycerol 3-phosphate acyltransferase, acyl-CoA:diacylglycerol acyltransferase, and acyl-CoA:acylglycerophosphocholine acyltransferase. The effect of ozone on lipid synthesis is closely comparable to the inhibition by N-ethylmaleimide suggesting that the effect of ozone is the oxidation of enzyme sulfhydryl groups. There was no indication of lipid oxidation caused by ozone and no indication of the production of a stable toxic compound.

The sensitivities of creatine and adenylate kinases to the neurotoxins acrylamide and methyl n-butyl ketone

It has recently been proposed that the similar multifocal axonopathies caused by several industrial toxins are due to the inhibition of metabolic enzymes with sensitive sulfhydryl groups. We find that the neurotoxins acrylamide and methyl n-butyl ketone in millimolar concentrations irreversibly inhibit rat brain and rabbit muscle creatine kinase and brain adenylate kinase. Brain creatine kinase was more sensitive to the toxins than was muscle creatine kinase or brain adenylate kinase. The chemically related but “nonneurotoxic” agents, N,N′-methylene bisacrylamide and methyl isobutyl ketone, were somewhat less effective than the known toxins. Dithiothreitol protected the enzymes against the toxins. However, differences in the effects of temperature and the protection by dithiothreitol indicated complex enzyme -toxin -dithiothreitol interaction. Protection by dithiothreitol does not necessarily implicate enzyme sulfhydryl groups or the site of toxin action. The lack of of cificity of toxin action and the concentrations of toxin required make it unlikely that metabolic enzyme inhibition alone accounts for hydrocarbon axonopathy.

Human aldehyde dehydrogenase: mechanism of inhibition of disulfiram.

Disulfiram labeled with carbon-14 reacts specifically with human liver aldehyde dehydrogenase E1 with loss of catalytic activity and no incorporation of label. Carbon-14-labeled diethyldithiocarbamate is formed and the number of enzyme sulfhydryl groups decreases from 34 to 30 during this process. Activity is recovered by-mercaptoethanol but not by glutathione, the physiological reducing agent.

Cystamine-Sepharose. A probe for the active site of gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase, previously known to be potently inhibited by cystamine, has been found to bind covalently to cystamine-Sepharose. ATP facilitates, whereas glutamate plus magnesium ions inhibit, binding of the enzyme to cystamine-Sepharose. A large fraction of the enzyme applied to columns of cystamine-Sepharose binds by forming a disulfide bond between cysteamine-Sepharose and a sulfhydryl group at or near the active site of the enzyme. The enzyme may be released by treatment with dithiothreitol. Some of the enzyme applied to such columns is inactivated and not bound covalently to the column. That the enzyme does not bind to columns of S-(S-methyl)cysteamine-Sepharose, whereas free S-(S-methyl)cysteamine is a potent inhibitor, indicates that a cysteamine-S disulfide moiety derived from the external cysteamine residue of cystamine-Sepharose is the critical group recognized by the enzyme. The observed partitioning of the enzyme on columns of cystamine-Sepharose between covalently column-bound enzyme and nonbound inactivated enzyme suggests that the reactive enzyme sulfhydryl group forms a disulfide linkage with the sulfur atom at the immobilized end of cystamine to link the enzyme to the column and to liberate free cysteamine, and also that the enzyme interacts with the external cysteamine moiety of the bound cystamine. The latter may occur if the free cysteamine released is spontaneously oxidized to free cystamine followed by its inhibition of the enzyme, or if there is a direct reaction between the enzyme-reactive sulfhydryl group and the sulfur atom of the external cysteamine moiety of cystamine-Sepharose.

Involvement of sulfhydryl groups in the oxidative modulation of particulate lung guanylate cyclase by nitric oxide and N-methyl- N′-nitro- N-nitrosoguanidine

Particulate guanylate cyclase from rat lung was activated by nitric oxide or if N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) in a dose-dependent manner that was enhanced by dithiothreitol. Nitric oxide-stimulated guanylate cyclase activity decayed during a 60-min preincubation at 37°, but did not decay at 24° or 4°. Dithiothreitol enhanced the decay of nitric oxide-stimulated enzyme at all temperatures by potentiating the reversal of nitric oxide activation. Following the reversal of nitric oxide activation at 24° by dithiothreitol, the particulate enzyme could be reactivated by a second exposure to nitric oxide. Preincubation of basal particulate guanylate cyclase activity at 37° resulted in the loss of enzyme responsiveness to activation by nitric oxide or MNNG that was potentiated by diamide or oxidized glutathione. The inhibitory effects of the thiol oxidants on enzyme responsiveness to activation by MNNG were prevented by dithiothreitol. The results suggest that activation of particulate guanylate cyclase by nitric oxide or MNNG involves the oxidation of key enzyme sulfhydryl groups.

Effect of chemical modification of sulfhydryl groups of human erythrocyte enzymes.

Erythrocyte lysate proteins by five methods were chemically modified by methanethiolation with methyl methanethiolsulfonate (MMTS). This relatively newly studied agent delivers the small uncharged, non-hydrogen-bonding CH3S-group to accessible -SH groups under mild conditions. Glycolytic and nonglycolytic red cell enzymes studied could be grouped into four categories based on degree of catalytic inhibition by MMTS, which ranged from 100% to nil. NADH ((0.06 mM) protected against lactate dehydrogenase (LDH) inhibition nearly completely, and partial protection was evident at 1 microM concentrations. NAD was virtually ineffective at comparable molarities, and pyruvate failed to protect significantly. Glucose-6-phosphate dehydrogenase (G-6-PD) (but not 6-PGD) was protected by NADP (but minimally if at all by NADPH) at low molarities. Substrate G-6-P failed to protect. Mg++ alone protected enolase to a major degree against MMTS inhibition. Residual activities of several enzymes were rendered highly unstable to thermal stress, and in the case of selected enzymes, alteration of pH activity curves and catalytic activity at diminished substrate concentration was demonstrable. The highly specific modification of sulfhydryl groups of cysteinyl residues under mild conditions preserving other aspects of enzyme structure provides insights into the role of these most reactive of all amino side-chains in the enzyme proteins derived from human red cell lysates.

Characterization of essential enzyme sulfhydryl groups of thyroxine 5'-deiodinase from rat kidney.

T4 5′-deiodinase, found in cell membranes of kidney and liver, is a thiol-dependent enzyme inhibited by propylthiouracil (PTU). Pretreatment of enzymatically active kidney membrane preparations (Mx) with PTU (10 μm) and increasing concentrations of L-T4 under N2 resulted in increasing degrees of persistent inhibition, as assayed in the absence of pretreatment reagents. Aerobic pretreatment with PTU in the presence or absence of L-T4 resulted in 80% inhibition of T4 5′-deiodination. Sulfhydryl modification of kidney Mx with iodoacetate or Nethylmaleimide irreversibly inactivated T4 5′-deiodinase. The formation of a reversible PTU-enzyme complex was found to protect T4 5′-deiodination from irreversible inactivation by iodoacetate or N-ethylmaleimide. A dose-dependent increase in the 35S content of kidney Mx was found after injection of [35S]thiouracil (TU) into rats, and the Mx 35S content was reduced 4-fold by simultaneous injection of methimazole. Treatment of kidney Mx, containing bound ;ISS, with 10 mm ...

Studies on regulatory functions of malic enzymes. VII. Structural and functional characteristics of sulfhydryl groups in NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme from Escherichia coli W contains 7 cysteinyl residues per enzyme subunit. The reactivity of sulfhydryl (SH) groups of the enzyme was examined using several SH reagents, including 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). 1. Two SH groups in the native enzyme subunit reacted with DTNB (or NEM) with different reaction rates, accompanied by a complete loss of the enzyme activity. The second-order modification rate constant of the "fast SH group" with DTNB coincided with the second-order inactivation rate constant of the enzyme by the reagent, suggesting that modification of the "fast SH group" is responsible for the inactivation. When the enzyme was denatured in 4 M guanidine HCl, all the SH groups reacted with the two reagents. 2. Althoug the inactivation rate constant was increased by the addition of Mg2+, an essential cofactor in the enzyme reaction, the modification rate constant of the "fast SH group" was unaffected. The relationship between the number of SH groups modified with DTNB or NEM and the residual enzyme activity in the absence of Mg2+ was linear, whereas that in the presence of Mg2+ was concave-upwards. These results suggest that the Mg2+-dependent increase in the inactivation rate constant is not the result of an increase in the rate constant of the "fast FH group" modification. 3. The absorption spectrum of the enzyme in the ultraviolet region was changed by addition of Mg2+. The dissociation constant of the Mg2+-enzyme complex obtained from the Mg2+- dependent increment of the difference absorption coincided with that obtained from the Mg2+- dependent enhancement of NEM inactivation. 4. Both the inactivation rate constant and the modification rate constant of the "fast SH group" were decreased by the addition of NADP+. The protective effect of NADP+ was increased by the addition of Mg2+. Based on the above results, the effects of Mg2+ on the SH-group modification are discussed from the viewpoint of conformational alteration of the enzyme.

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.

The Interactions of adenylates with allosteric citrate synthase

Evidence is presented that a number of derivatives of adenylic acid may bind to the allosteric NADH binding site of Escherichia coli citrate synthase. This evidence includes the facts that all the adenylates inhibit NADH binding in a competitive manner and that those which have been tested protect an enzyme sulfhydryl group from reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) in the same way that NADH does. However, whereas NADH is a potent inhibitor of citrate synthase, most of the adenylates are activators. The best activator, ADP-ribose, increases the affinity of the enzyme for the substrate, acetyl-CoA, and saturates the enzyme in a sigmoid manner. A fluorescence technique, involving the displacement of 8-anilino-1-naphthalenesulfonate from its complex with citrate synthase, is used to obtain saturation curves for several nucleotides under nonassay conditions. It is found that acetyl-coenzyme A, coenzyme A, and ADP-ribose all bind to the enzyme cooperatively, and that the binding of each becomes tighter in the presence of KCl, the activator, and oxaloacetic acid (OAA), the second substrate. Another inhibitor, alpha-ketoglutarate, can complete with OAA in the absence of KCl but not in its presence. The nature of the allosteric site of citrate synthase, and the modes of action of several activators and inhibitors, are discussed in the light of this evidence.

Inhibition of malate dehydrogenase by thyroxine and structurally related compounds

The inhibition of pig heart mitochondrial malate dehydrogenase ( l-malate: NAD + oxidoreductase, EC 1.1.1.37) by thyroxine and structurally related compounds was studied to resolve a longstanding question about the exact nature of the inhibition. Thyroxine, in freshly prepared solution, was found to be a “pure” conpetitive inhibitor relative to the nucleotide cofactor. Upon standing in diffuse daylight, solutions of thyroxine showed increased ability to inhibit the enzyme, presumably as a result of oxidation of enzyme sulfhydryl groups by free iodine that is released photochemically. This behavior probably accounts for earlier reports of irreversible inactivation by thyroxine. Comment is made on the implications of these findings to the mechanism of thyroid hormone action.

Inhibition of γ-glutamylcysteine synthetase by cystamine; An approach to a therapy of 5-oxoprolinuria (pyroglutamic aciduria)

γ-Glutamylcysteine synthetase is strongly inhibited by cystamine; thus, 20 μM cystamine inhibited the activity by 50%. Inhibition is rapid and the inhibited enzyme is reactivated by dithiothreitol suggesting that cystamine reacts with an enzyme sulfhydryl group. Inhibition by cystamine is not prevented by MgATP, L-α-aminobutyrate, or L-glutamate suggesting that cystamine may not interact at the active site. Little or no inhibition was observed with N,N′-diacetyl cystamine, L-cystine, glutathione disulfide, 2-hydroxyethyl disulfide, and thioglycolate disulfide, whereas thiocholine disulfide produced moderate inhibition. Cystamine or an inhibitory analog of cystamine might be useful in the therapy of the disease 5-oxoprolinuria in which there is an overproduction of γ-glutamylcysteine.

Sulfhydryl group reactivity in the Escherichia coli CoA transferase

The acetyl-CoA:acetoacetate CoA-transferase of Escherichia coli has the subunit structure α 2 β 2 The enzyme contains six sulfhydryl groups, one per α chain and two per β chain, and no disulfides. The rates and extent of sulfhydryl group reactivity with 5,5′-dithiobis(2-nitrobenzoic acid) were compared in the free enzyme, the enzyme-CoA intermediate in the catalytic pathway, and a substrate analog-enzyme Michaelis complex. The analog used was acetylaminodesthio-CoA, a competitive inhibitor with respect to acetyl-CoA; the analog is not a substrate. The reactions were studied in the presence and absence of 10% glycerol. In the absence of glycerol, one sulfhydryl group reacted rapidly in the free enzyme and enzyme-CoA intermediate; relative to the free enzyme, the rate and number of subsequently reacting sulfhydryl groups were increased in the enzyme-CoA intermediate. In the presence of 10% glycerol, one sulfhydryl group reacted rapidly in the free enzyme, while two reacted rapidly in the enzyme-CoA compound; the rates and extents of subsequently reacting sulfhydryl groups were also enhanced in the enzyme-CoA compound. The data strongly suggested subunit interactions in the free enzyme and intermediate; glycerol abolished those interactions in the enzyme-CoA intermediate. In the absence of glycerol, sulfhydryl group reactivity in the Michaelis complex, enzyme-acetylaminodesthio-CoA, was similar to that in the free enzyme with one exception: One of the more slowly reacting sulfhydryl groups in the free enzyme reacted at a rate characteristic of the enzyme-CoA intermediate. The results obtained with N-ethylmaleimide were qualitatively similar. The fractional inactivation of the enzyme with N-ethylmaleimide as a function of sulfhydryl groups modified and the subunit location of those sulfhydryl groups indicated that the same sulfhydryl groups react in both enzyme species; however, those sulfhydryl groups reacted more rapidly in the enzyme-CoA compound. The data indicate both subunit interactions in the enzyme and characteristic conformational changes upon formation of an acyl-CoA-enzyme Michaelis complex and the enzyme-CoA intermediate.

The effects of sulfhydryl reducing and modifying agents on aminoacyl-tRNA synthetases from calf liver

The effect of various sulfhydryl reagents on thein vitro activity of calf liver aminoacyl-tRNA synthetases has been tested. With few exceptions reduction or alkylation of enzyme sulfhydryl groups lead to a loss of enzyme activity. Further, the sensitivities to reducing and mercaptide forming reagents are apparently related.