Reactive SH Groups Research Articles

Acetylcholine receptor: SH group reactivity as indicator of conformational changes and functional states.

Acetylcholine receptor: SH group reactivity as indicator of conformational changes and functional states.

Sulfhydryl groups in relation to the structure and catalytic activity of 2-OXO-4-hydroxyglutarate aldolase from bovine liver

Bovine liver 2-oxo-4-hydroxyglutarate aldolase (suggested name: 2-oxo-4hydroxyglutarate glyoxylate-lyase catalyzing the reaction: 2-oxo-4-hydroxyglutarate rlhar2 pyruvate + glyoxylate) contains eight to ten sulfhydryl groups as determined by titration of the enzyme with either 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs 2) or p-mercuribenzoate in the presence of 1% sodium dodecyl sulfate. In the absence of a denaturant, all of the cysteinyl residues react with p-mercuribenzoate whereas only four are accessible to titration with Nbs 2. No differences in -SH group reactivity can be detected during titration of the aldolase with p-mercuribenzoate. In contrast, two classes of sulfhydryls can be differentiated in the disulfide exchange reaction with Nbs 2 in the absence of a denaturant; one -SH group (Class I) reacts rapidly whereas three additional thiols (Class II) titrate at approx. 0.1 the rate of the Class I -SH residue. Both pyruvate and glyoxylate protect one of the three -SH residues in Class II from reaction with Nbs 2. Either substrate also prevents titration of one to two thiol groups by p-mercuribenzoate and decreases the rate of reaction of aldolase -SH groups with Nbs 2 in 8 M urea. These ligand-induced changes in -SH reactivity provide a sensitive indication that the enzyme exists in an altered conformational state in the presence of either of its cosubstrates. Titration of the enzyme with either Nbs 2 or p-mercuribenzoate results in a progressive loss of aldolase activity which is not proportional to the number of -SH groups modified. The enzyme retains 50% of the activity of the native enzyme when Class I and Class II thiols (i.e. four -SH groups total) are modified with Nbs 2; 15% residual activity is still observed following titration of all of the cysteinyl residues with p-mercuribenzoate. Pyruvate and glyoxylate provide partial protection against inactivation. It is concluded that inactivation of 2-oxo-4-hydroxyglutarate aldolase by Nbs 2 or p-mercuribenzoate is a consequence of alterations in protein structure which accompany modification of -SH groups. The data argue against the direct participation of an active-site thiol group in the catalytic mechanism of 2-oxo-4-hydroxyglutarate aldolase, be that aldol cleavage and condensation or β-decarboxylation.

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.

Rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase: Adenine nucleotide-induced inactivation

Glyceraldehyde-3-phosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12), isolated from rat skeletal muscle undergoes a rapid inactivation upon incubation at 25 °C in the presence of adenine nucleotides. The reaction can be described as a reversible tetramerdimer equilibrium, only the tetrameric form of the enzyme being active in the presence of nucleotides. The standard free energy changes upon dissociation at 25 °C in 0.1 m phosphate buffer pH 7.5 in the presence of saturating concentrations of ATP, ADP, AMP, and ADP-ribose were found to be 6.69, 6.93, 8.31, and 10.5 kcal/mol, respectively. Nucleotide-dependent inactivation does not bring about any alteration of the reactivity of SH groups of the enzyme towards 5,5′-dithiobis(2-nitrobenzoic acid). This is not the case, however, when the enzyme undergoes NaCl-induced cold inactivation, which is accompanied by an increased accessibility of SH groups. ADP and ATP protect the enzyme against cold inactivation in the presence of NaCl and decrease the enhanced reactivity of SH groups. Adenine nucleotide-induced inactivation is prevented in the presence of NAD. The protective effect is noncooperative, the extent of inactivation being dependent upon the amount of active centers free of bound coenzyme. Addition of excess NAD to the inactivated enzyme results in a complete regain of activity. A comparative study made on the rate of reforming enzyme NAD complex (followed spectrophotometrically) and the regain of activity has demonstrated that the former process is markedly more rapid than the latter. The reactivation was observed to follow second-order kinetics, which suggests that the reassociation of the inactive NAD-liganded dimers is the rate-limiting step. The data are consistent with the existence of different conformational transitions responsible for the restoration of the intersubunit contact area, catalytic activity, and thermal stability of the enzyme molecule, respectively.

The effect of ATP upon the reactivity of SH groups in sarcoplasmic reticulum membranes

The effect of ATP upon the reactivity of SH groups in sarcoplasmic reticulum membranes

Microheterogeneity in porcine pancreatic amylase preparations due to disulfide-sulfhydryl exchange

There are two masked SH groups in pancreatic amylase (EC 3.2.1.1) which become reactive after removal of Ca ions, and then only due to fluctuation of the polypeptide chain. Carboxymethylation of the masked SH groups and isolation of the tryptic CM-peptides have shown that the reacting SH groups in active amylase preparations are not identical, depending on the method of preparation. The microheterogeneity is attributed to an SH-SS exchange reaction taking place during preparation. This exchange results in a mixture of two types of amylase molecules containing different SH groups. The tryptic peptides containing the SH groups have been detected from the r radioautogram of the peptide map of the [ 14C] carboxymethylated protein digest. We identified the SH peptides present in native amylase, and those cysteinyl peptides which form an wasily reducible disulfide in the vicinity of these thiol groups, and take part in the intramolecular SH-disulfide exchange.

Distribution of free sulfhydryl and disulfide groups among platelet membrane proteins

Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet membranes treated with N- ethyl[1− 14 C] maleimide , phenyl[ 203Hg]mercuric acetate and p- chloro[ 203 Hg] mercuribenzoate showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from > 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with 14C-labeled 5,5′-dithio-bis(2-nitrobenzoic acid) resulted in changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon 5,5′-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.

Comparative studies on reactivity of sh groups of chromatin, histones and low-molecular weight thiols

1. 1. Kinetics of reactions of SH groups of calf thymus chromatin, total histone (T) and histone F3 with 5,5'-dithiobis (2-nitrobenzoate), (DTNB) and β-hydroxyethyl-2,4-dinitrophenyl disulphide (DNP-HED) was determined and compared with those of reduced glutathione, cysteine and 2-mercaptoethylamine. 2. 2. Chromatin SH groups were found to react with uniform first order kinetics. 3. 3. Time courses of reactions of histone F3 and T with both disulphides have been found to be biphasic. About 80% of SH groups of histone F3 or T was found to react extremely fast. The remaining 20% reacted considerably slower, according to first order kinetics. 4. 4. It may be presumed that two types of SH groups of histone F3 presented in this study come about as a result of its microheterogenity noted by Marzluff et al. (1972) and Patthy & Smith (1975).

Involvement of sulfhydryl groups in the action of insulin and radiation on thymocyte Na +-dependent amino acid transport

1. 1. p- Chloromercuribenzene sulfonate concentrations <10 −5 M stimulate the the uptake by thymocytes of 2-aminoisobutyrate, a non-metabolized amino acid. At concentrations > 10 −5 M of this reagent, transport is impaired and cell viability is affected. In contrast, 5,5′-dithiobis-(2-nitrobenzoate) between 10 −4 and 10 −6 M produces only stimulation of 2-aminoisobutyrate uptake after treating for 10 min. 2. 2. Treatment of thymocytes with 10 −4 M 5,5′-dithiobis-(2-nitrobenzoate) reveals at least three categories of reactive SH groups. Titration of the most rapidly reacting category, 4 · 10 7−7 · 10 7/ cell , activates 2-aminoisobutyrate transport to the same extent as does p- chloromercuribenzene sulfonate. Cells treated with 10 −6 M insulin showed a 30–50% reduction in the number of sulfhydryl groups that could be titrated with 5,5′-dithiobis-(2-nitrobenzoate). In thymocytes treated with 10 −6 M p- chloro[ 203Hg] mercuribenzene sulfonate, addition of 10 −6 or 10 −9 M insulin before treatment with the sulfhydryl reagent again reduces the number of titrable SH groups by 20%. 3. 3. Insulin (10 −10−10 −6 M) also stimulates 2-aminoisobutyrate uptake, but the effects of insulin and SH blocker are not addictive. 4. 4. Insulin, but not p- chloromercuribenzene sulfonate, prevents the impairment of 2-aminoisobutyrate transport caused by γ-irradiation. Treatment of cells with p- choloromercuribenzene sulfonate prior to irradiation increases the radiation impairment of 2-aminoisobutyrate transport. 5. 5. γ-irradiation reduces the number of 5,5′-dithiobis-(2-nitrobenzoate) reactive sulfhydryl residues by 37%. 6. 6. A model for the action of insulin and irradiation on 2-aminoisobutyrate transport is presented.

Bestimmung von Sulfhydryl und Disulfid-Gruppen in Proteinen mit Hilfe der amperometrischen Titration

The specifity of Ag+ ions for protein SH groups has been questioned frequently, even though the amperometric titration with AgNO3 is one of the most common methods for the determination of SH groups in proteins. This is due to the fact, that the formation of silver complexes in the titration of cysteine causes a consumption of AgNO3 which is too high. In order to find out if this may be true in the case of proteins, in the present work select proteins with a well known content of SH and SS groups have been titrated amperometrically in tris buffer pH 7.4 with 0.001 M AgNO3. The proteins used were hemoglobin, bovine serum albumin, ovalbumin, lysozyme, pepsin, myoglobin, and cytochrome c. The direct and the indirect titrations of (a) native, (b) denatured, and (c) NaBH4 reduced proteins showed, that the expected consumption of AgNO3 was in no case exceeded. Therefore under the conditions used AgNO3 may be considered as a specific reagent for protein SH groups. High SH values as a result of the amperometric titration of proteins with silver nitrate, which have been published occasionally, may be due to incorrect estimation of the end point of the titration. The reducibility of SS groups depends on the kind of protein. Lysozyme and pepsin were already completely reduced at 23 degrees C, whereas bovine serum albumin needed 60 degrees C. The direct titration method was useful only in some cases for the detection of all SH groups originally present in the proteins or formed by reduction with NaBH4. On the other hand the indirect titration method gave maximum values, because the slowly reacting SH groups of proteins are also allowed to react and the resulting titration curves may be evaluated correctly.

Effect of phosphate and ionophores on [ 14C]-NEM incorporation in mitochondrial membranes and relationships with phosphate carrier system

Phosphate transport in mitochondria was investigated with respect to its inhibition by NEM. The reactivity of the Pi carrier SH groups was influenced by phosphate or ionophores during preincubation before the addition of NEM. Furthermore in order to obtain some mitochondrial protein fractions where the typical effects of phosphate and ionophores on [14C]-NEM fixations were observed, mitochondria were submitted to hypotonic treatment and sonication. The following results were obtained: 1. -- Phosphate and grisorixin (a new ionophore of the nigericin group) decreased the inhibition of phosphate transport by NEM. The same effect was observed for [14C]-NEM incorporation. 2. -- Valinomycin increased [14C]-NEM incorporation. The valinomycin effect was abolished by phosphate. ClCCP alone affected [14C]-NEM incorporation slightly. Valinomycin plus ClCCP decreased NEM inhibition of phosphate transport and [14C]-NEM incorporation like grisorixin. 3. -- The variability of SH group reactivity can be interpreted by a control of SH group accessibility by transmembrane delta pH as previously suggested. 4. -- Typical effects of phosphate or ionophores were observed in whole pig heart and rat liver mitochondria. These effects were enhanced in the same supernatant protein fraction resulting from sonication in pig heart mitochondria : phosphate decreased [14C]-NEM incorporation by 1,50 nmoles/mg protein, grisorixin by 0.95 nmoles, whereas valinomycin increased it by 0.75 nmoles. For rat liver mitochondria the phosphate effect and the valinomycin increased it by 0.75 nmoles. For rat liver mitochondria the phosphate effect valinomycin effect on [14C]-NEM incorporation were observed in the subparticular fraction obtained after sonification.

The nature of inactivation of rat muscle 5'-adenylate aminohydrolase by fluorodinitrobenzene

1. The inactivation of rat skeletal muscle AMP deaminase by Dnp-F (1-fluoro-2,4-dinitrobenzene) is accompanied by the arylation of thiol, amino and phenolic hydroxyl groups. 2. The number of thiol groups that react with Dnp-F is about 12; this is the number that reacts with Nbs2 [5,5'-dithiobis-(2-nitrobenzoic acid)] and N-ethylmaleimide without loss of enzyme activity, and it appears to be the same thiol groups that all three reagents attack. 3. Dinitrophenylation of these reactive SH groups is not the cause of inactivation, since active N-ethylmaleimide-substituted enzyme is also inactivated by Dnp-F.4. Complete inactivation of the N-ethylmaleimide-treated AMP deaminase occurs when about six tyrosine and two lysine residues are dinitrophenylated. 5. Since the treatment of Dnp-enzyme with 2-mercaptoethanol restores much of the enzyme activity, inactivation of AMP deaminase by Dnp-F is probably largely due to modification of tyrosine residues. 6. The kinetic properties of the Dnp-enzyme indicate that a marked decrease in V occurs only after extensive enzyme modification. The decreased activity after slight inactivation results from modification of Km.

Properties of the highly reactive SH groups of phosphorylase b.

The reaction of rabbit muscle phosphorylase b with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied with stopped-flow spectrophotometry. Two highly reactive sulfhydryl groups per dimer reacted with Nbs2 within a few seconds, while the remaining SH groups needed several minutes and hours. Decomposition of the time curve revealed that the highly reactive SH groups can be divided into two subclasses: a fast type which reacted with a rate constant of 3 x 10-3 M-1 sec-1 and a more slowly reacting type disappearing with a rate constant of 0.3 x 10-3 M-1 sec-1. The reactivity of the slowly reacting type increased by a factor of about 2 in the presence of 1 mM AMP. Concurrently, the ratio between the fast reacting and the more slowly reacting subclasses decreased from 5.3 to 1.0. The AMP effect was greatly enhanced by glucose 1-phosphate. This enhancement was abolished in the presence of ATP. The finding that the ratio between the number of SH groups in the two subclasses of the highly reactive SH groups changed upon addition of ligand molecules indicates that the two subclasses reflect the different reactivities of the SH groups when the enzyme is present in different conformational states. It is suggested that the highly reactive SH group measured belong to the peptide: Gly-Cys-Arg-Asp.

On the structure of putidaredoxin and cytochrome P-450 cam and their mode of interaction.

Structural and immunochemical experiments with putidaredoxin, cytochrome P-450cam, and their 1:1 complex have led us to the following conclusions: Despite the remarkable sequence homology between putidaredoxin and adrenodoxin which permits a tentative assignment of cysteines binding to the (Fe-S)2 prosthetic group, these redox proteins cannot replace each other in reconstitution experiments because putidaredoxin contains a disulfide loop close to its P-450cam binding site. This feature may also be responsible for the complete lack of immunochemical cross reactivity between these proteins. The stability of putidaredoxin can be enhanced significantly by cross linkage with glutaraldehyde without change in spectral, catalytic, or immunochemical properties, Putidaredoxin also gains stability by binding to the P-450-camphor complex in a 1:1 ratio. Precipitation of this complex with anti-P-450cam antibodies gives access to site specific antibodies directed against the putidaredoxin binding site of P-450cam. A series of putidaredoxin-cytochrome P-450cam-substrate complexes with ratios of 1 to 6 molecules of redoxin per molecule of cytochrome have been obtained by migration of excess redoxin across prefocused P-450cam in electrofocusing. Complete inhibition of camphor hydroxylation was achieved by anti-P-450cam antibodies, their Fab fragments, anti-putidaredoxin-trimer antibodies, and antibodies directed against the putidaredoxin-P-450cam complex. Five major antigenic sites were tentatively established for P-450cam, two of which seem to be associated with the BrCN hemepeptide while one each relates to the putidaredoxin binding site, the Trp-Arg site close to the C-terminus, and the site surrounding the most reactive SH group which gives rise to dimer formation. Iodination, of P-450cam at tyrosyl residues only permitted use of a sensitive radioimmunoassay procedure for testing of cross reacting material (CRM) remaining after degradation of P-450cam with BrCN and enzymes, denaturation with acetone, and complex formation with the redoxin. The BrCN hemepeptide still has a Soret maximum at 390 nm and reacts with CO yielding a P-420 spectrum. All 6 half-cystines of P-450cam are present as free sulfhydryls and can be titrated after denaturation but only 4 of them are available in the P-450-camphor complex. Three of these are close to each other and the heme, and work in concert; their alkylation with N-ethyl maleimide (NEM) leads to shifts of the Soret from 391 to 417 nm and concomitant changes in redox potential, EPR-signals and DPNH-reactivity. The fifth SH group is protected by camphor while the 6th SH group, still present in the BrCN heme-peptide, is implicated in chelation to the heme iron by a drastic change in EPR spectra, reflecting pure axial symmetry at the heme after complete alkylation by NEM.

Polymerization of oxygenated and deoxygenated bull-frog hemoglobins

1. 1.|The adult bullfrog oxyhemoglobin polymerized to form the 7-S and 11-S molecules when stored in vitro. The reaction was accompanied by the loss of two reactive SH groups per hemoglobin tetramer, and was completely prevented by the treatment of hemoglobin with iodoacetamide. These observations agree essentially with those reported by other authors for the bullfrog hemolysate which was saturated with CO. 2. 2.|The adult bullfrog hemoglobin was found also to polymerize to a 7-S molecule when deoxygenated. The reaction was rapid and reversible with respect to the oxygenation-deoxygenation of the hemoglobin molecule. No reactive SH groups appear to be involved in this polymerization, because iodoacetamide and mercaptoethanol could not prevent the reaction. 3. 3.|The organic phosphates had no effect on the deoxy polymer formation of the stripped bullfrog hemoglobin. 4. 4.|Neither oxy- nor deoxyhemoglobin of the tadpole showed any tendency to polymerize under the same conditions in which the adult hemoglobin polymerized.

Changes in sulfhydryl groups of honeybee glyceraldehyde phosphate dehydrogenase associated with generation of the intermediate plateau in its saturation kinetics.

Honeybee and rabbit muscle GPDH were studied to obtain information at the chemical level regarding anomolous saturation kinetics of the honeybee enzyme. Results demonstrate that the enzyme's sulfhydryl groups are implicated in the process. Measured by DTNB titration, native honeybee GPDH has one less active SH than the native rabbit muscle enzyme and displays changes in overall sulfhydryl reactivity after preincubation with G-3-P or G-3-P plus NAD+. The total DTNB reactive sulfhydryls of rabbit muscle GPDH are not changed by preincubation with NAD+ or G-3-P; honeybee GPDH, under certain conductions of preincubation with these ligands, shows a decrease of two total DTNB reactive SH groups. This difference has been confirmed by an independent experiment in which the two enzymes were carboxymethylated with C-14 bromoacetic acid.

Haemoglobin, a sulphhydryl-protein in the binding reaction with radioprotective MEG.

SummaryHaemoglobin (Hb) in the form of oxyhaemoglobin as a SH protein binds SH radioprotector MEG through its reactive SH-groups. Prolonged reaction time, alkaline pH and urea are able to enhance the reactivity of the unreactive (‘masked’) SH-groups of Hb to the radioprotector. The binding of MEG to the β-93 SH-groups of Hb increases the chromatographic and electrophoretic heterogeneity of the macromolecule.On 60Co γ-irradiation, the mixed disulphide bond between Hb and MEG is not broken but stabilized, thus provoking structural changes, resulting in denaturation. The denatured protein retains the firmly bound radioprotector; the mixed disulphide bond resists splitting with thiols after being irradiated.

Study of correlation between structural motility and reactivity of SH groups in α-amylase

1. 1. The two masked SH groups of pancreatic α-amylase (α-1,4-glucan 4-glucano-hydrolase, EC 3.2.1.1) react with 5,5′-dithio-bis(2-nitrobenzoate) following first-order kinetics, when EDTA is present. Since the reaction is not accelerated on prolonged preincubation of amylase with EDTA, local fluctuation of the polypeptide chain is supposed to be the rate-limiting step of the reaction 2. 2. As a result of this reaction of the two SH groups, either one intramolecular, or two mixed disulfides are formed, depending on the relative concentrations of the enzyme and 5,5′-dithio-bis(2-nitrobenzoate). 3. 3. In 7 M urea one SH group becomes unmasked due to the partial unfolding, while the reaction of the other SH group is still limited by fluctuation of the relevant part of the protein molecule. 4. 4. Oxidation of SH groups by 5,5′-dithio-bis(2-nitrobenzoate), or the formation of mixed (enzyme-2-nitro-5-mercaptobenzoate) disulfide does not influence the enzyme activity, but decreases its stability.

SH groups masked by subunit interaction in glyceraldehyde- 3-phosphate dehydrogenase

The changes in reactivity of the less reactive SH groups of glyceraldehyde-3-phosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating) EC 1.2.1.12) apoenzyme towards thiol reagents were studied during the course of dissociation into dimers and inactivation of the enzyme induced by stoichiometric amounts of ATP. Neither the reactive SH group of Cys-149 nor that of Cys-153 (the latter being the second cysteinyl group in order of reactivity towards thiol reagents) is ozidized during dissociation and inactivation caused by ATP treatment. However, during this process one SH group per subunit which was previously masked in the native tetramer is oxidized. This cysteinyl residue corresponds to Cys-281 as identified anallytically. The molecular weight of the ATP-treated enzyme is 60 000 even in the presence of 6 M urea. Both enzymatic activity and the oxidized SH group can be fully recovered by treatment with 2-mercaptoethanol. We conclude that during dissociation into dimers by ATP, residues Cys-281 form interchain S-S bridges within the dimers.

Differential reactivity of the sulfhydryl groups of yeast aldolase toward p-hydroxymer curibenzoate

Reaction of p-hydroxymercuribenzoate with sulfhydryl groups in yeast aldolase was studied by the stopped-flow method. Two classes of SH groups can be distinguished. Fast reactive SH groups (approximately 3) have the rate constant around 4 × 10 5 M −1sec −1, which is at least 10 3 faster than the slow reactive SH groups (approximately 2 in holoenzyme). Apoaldolase has 2 additional slow reactive SH groups. Fructose diphosphate blocks the reaction of at least one slow reactive SH group both in holo- and apo-enzymes. These results suggest that the SH groups of yeast aldolase are involved in the fructose diphosphate binding site and possibly involved as a ligand on the crucial Zn atom.