The Number, Location, and Reactivity of the Cysteine Residues of Sturgeon Muscle Aldolase

1972 ◽  
Vol 50 (2) ◽  
pp. 111-119 ◽  
Author(s):  
P. J. Anderson
Keyword(s):  
Iodoacetic Acid ◽  
Sulfhydryl Groups ◽  
Cysteine Residues ◽  
Rabbit Muscle ◽  
Nitrobenzoic Acid ◽  
Catalytic Role ◽  
Aldolase Activity ◽  
Activity Loss

Sturgeon muscle aldolase contains six cysteine residues per subunit. These residues appear to occur in homologous positions to six of the eight cysteine residues of rabbit muscle aldolase. Three of the six residues can react with either iodoacetic acid or 5,5′-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. Reaction of three residues with iodoacetic acid inactivates the enzyme. The presence of substrate protects one of these residues and in this case no activity loss is observed. However, the results indicate that the effects on activity are due to the addition of the modifying group rather than to a loss of sulfhydryl groups. No residue corresponding to the previously demonstrated substrate-protected cysteine of rabbit aldolase was located in the sturgeon enzyme, which demonstrated that this residue was not essential to aldolase activity. It therefore appears unlikely that cysteine residues have a direct or an auxiliary catalytic role in aldolase activity.

The sulfhydryl groups of muscle phosphorylase. I. Number and reactivity

1968 ◽  
Vol 46 (6) ◽  
pp. 609-615 ◽  
Author(s):  
M. L. Battell ◽  
L. B. Smillie ◽  
N. B. Madsen
Keyword(s):  
Glycogen Phosphorylase ◽  
Monomer Unit ◽  
Sodium Dodecyl ◽  
Spectrophotometric Titration ◽  
Iodoacetic Acid ◽  
Enzymic Activity ◽  
Sulfhydryl Groups ◽  
Rabbit Muscle ◽  
Complete Inactivation ◽  
Protein Sulfhydryl Groups

The sulfhydryl groups of glycogen phosphorylase from rabbit muscle have been investigated with respect to their reactivity with p-chloromercuribenzoate (PCMB), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), cystine, N-ethylmaleimide (NEM), iodoacetic acid, and iodoacetamide. By spectrophotometric titration, PCMB reacts with 6 of the 18 half-cystine residues in native phosphorylase b, and with 12–14 of the 36 half-cystine residues in native phosphorylase a, and causes complete inactivation in both cases. Reaction of approximately 6 more residues in phosphorylase b (12 in phosphorylase a) occurs when the protein is denatured with HCl or sodium dodecyl sulfate (SDS).DTNB, cystine, and iodoacetic acid react with only two sulfhydryl groups in native phosphorylase b, and do not cause any loss of activity. Iodoacetamide can also react with two sulfhydryl groups without causing any loss of enzymic activity, but activity is lost in proportion to the extent of titration of the next four groups. DTNB reacts with 12 groups in SDS-denatured phosphorylase b but with only 9 groups in urea- or guanidine HCl-denatured enzyme. NEM and iodoacetic acid react with 14 groups in urea-denatured phosphorylase b. Two experiments are reported that provide additional confirmation of previous reports of the absence of disulfide bridges in phosphorylase.We conclude that two sulfhydryl groups per mole of phosphorylase b can react with any reagent without loss of enzymic activity. When the correct reagent and conditions are chosen, a further class of four sulfhydryl groups reacts, resulting in complete loss of enzymic activity. Thus, only two specific sulfhydryl groups on each monomer unit need be titrated to inactivate glycogen phosphorylase and cause it to dissociate. Upon denaturation of phosphorylase by several of the usual protein denaturants, another class of sulfhydryl groups reacts readily, the exact number depending on the reagent and the conditions, but the complete titration of all 18 sulfhydryl groups per molecule of phosphorylase b occurs only under special conditions. These results are of general relevance to procedures employing various protein denaturants and sulfhydryl reagents for the titration of protein sulfhydryl groups.

scholarly journals Bovine inositol monophosphatase. Modification, identification and mutagenesis of reactive cysteine residues

1992 ◽  
Vol 285 (2) ◽  
pp. 461-468 ◽  
Author(s):  
M R Knowles ◽  
N Gee ◽  
G McAllister ◽  
C I Ragan ◽  
P J Greasley ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Iodoacetic Acid ◽  
Native Enzyme ◽  
Steric Effects ◽  
Cysteine Residues ◽  
Native Protein ◽  
Protein Fluorescence ◽  
Inositol Monophosphatase ◽  
Nitrobenzoic Acid ◽  
Full Activity

1. Bovine inositol monophosphatase reacts with thiol reagents such as 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM) and iodoacetic acid (IAA). 2. Modification by NEM results in nearly total loss of enzyme activity, whereas modification by IAA causes a slight increase in activity. 3. The loss of activity caused by NEM can be prevented by the inclusion of Ins1P, or better Ins1P and LiCl in the reaction mixture. 4. Two equivalents of p-nitrothiobenzoate (NTB2-) are released from the native enzyme on reaction with DTNB, and six equivalents of NTB2- are released from the SDS-denatured enzyme, suggesting that none of the six cysteine residues per molecule of enzyme is involved in intra- or inter-molecular disulphide bridges. 5. Both NEM and IAA react with two cysteine residues (residues 141 and 184 in the sequence) in a mutually exclusive manner. 6. NEM also reacts stoichiometrically with residue 218. 7. The NEM-induced loss of enzyme activity is accompanied by a 15% decrease in protein fluorescence. 8. A mutant of the enzyme which has an Ala-218 replacement for Cys-218 has full activity and is not sensitive to NEM, showing that the modification of this cysteine by NEM causes inhibition of the native protein by steric effects and that Cys-218 is not essential for activity.

scholarly journals Carboxymethylation of Thiol Groups in Ovalbumin: Implications for Proteins that Contain Both Thiol and Disulfide Groups

1982 ◽  
Vol 35 (2) ◽  
pp. 125 ◽  
Author(s):  
DM Webster ◽  
EOP Thompson
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Iodoacetic Acid ◽  
Cysteine Residues ◽  
Alkaline Ph ◽  
Thiol Groups ◽  
Nitrobenzoic Acid ◽  
Dodecyl Sulfate ◽  
Fold Excess

The cysteine residues of hen ovalbumin were S-carboxymethylated with non-radioactive iodoacetic acid under various conditions by altering the pH at which the protein was denatured in 8 M urea, by using different molar ratios of non-radioactive iodoacetic acid to cysteine and by varying the time at which carboxymethylation was commenced after denaturing conditions had been applied. Under the various conditions, the thiol groups were carboxymethylated to different extents, the residual thiol groups being measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecyl sulfate. When ovalbumin is carboxymethylated in alkaline urea, it unfolds slowly and the carboxymethylation is incomplete even with 150-fold excess iodoacetic acid. The known rapid thiol-disulfide exchange that occurs at alkaline pH values makes this method of carboxymethylation unsuitable as a preliminary step for blocking the native cysteine residues of ovalbumin before reduction and labelling the thiol groups formed by reduction of the disulfide bonds. Titration of the thiol groups of ovalbumin in 6 M guanidine hydrochloride or 1 % (w/v) sodium dodecyl sulfate at pH 8�2 with 5,5' -dithiobis(2-nitrobenzoic acid) is more rapid than in 8 M urea and these solvents would be preferable for studies of the disulfide-bonded sequences. Denaturation of ovalbumin in acidic 8 M urea is a very rapid process, and under mild acid conditions thiol-disulfide interchange is much slower. Subsequent carboxymethylation of the cysteine residues at alkaline pH with 150-fold excess iodoacetic acid results in complete carboxymethylation and the carboxymethylated ovalbumin can be reduced and labelled with radioactive iodoacetic acid with specific labelling of the half-cystine residues involved in the disulfide bond. The results are discussed in relation to the allocation of half-cystine residues in other protein systems that contain both thiol and disulfide groups.

scholarly journals The accessibility of the thiol groups on G- and F-actin of rabbit muscle

1990 ◽  
Vol 266 (2) ◽  
pp. 453-459 ◽  
Author(s):  
D F Liu ◽  
D Wang ◽  
A Stracher
Keyword(s):  
Thiol Group ◽  
Iodoacetic Acid ◽  
Drastic Change ◽  
The Other ◽  
Cysteine Residues ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Filamentous Actin ◽  
Other Hand

The accessibility of the cysteine residues of actin from rabbit muscles to the thiol-targeted reagent 7-dimethylamino-4-methyl-(N-maleimidyl)coumarin (DACM) was investigated. Under conditions where the actin is in the unpolymerized form (G-actin), the most reactive thiol group was Cys-257, suggesting that it was located on the surface of the actin molecule. The selective modification of Cys-374 for this reagent as reported by Sutoh [(1982) Biochemistry 21, 3654-3661] was not observed. Cys-10, Cys-217 and Cys-374 were much less reactive and only gradually became extensively modified when the concentration of DACM approached 5 molar equivalents of actin. Presumably these thiol groups were located further inward away from the surface or situated in a different environment that rendered them less reactive. On the other hand, Cys-285 was completely inaccessible and presumably was buried. The lack of preferential labelling of Cys-374 by DACM is incompatible with the finding with iodoacetic acid as the reagent as reported by Elzinga & Collins [(1975) J. Biol. Chem. 250, 5897-5905]. This discrepancy, however, might well be due to the different reagents employed. The DACM-G-actin largely retained its competence for polymerization. Upon polymerization of G-actin, practically all the thiol groups became inaccessible to DACM, suggesting that a drastic change occurred in the conformation of actin units in the transition of monomers to filamentous actin.

scholarly journals The reactivity of thiol groups and the subunit structure of aldolase

1970 ◽  
Vol 117 (2) ◽  
pp. 291-298 ◽  
Author(s):  
P. J. Anderson ◽  
R. N. Perham
Keyword(s):  
Group Analysis ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Native Enzyme ◽  
Cysteine Residues ◽  
Subunit Structure ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Prolonged Incubation ◽  
Nitrobenzoic Acid

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-14C]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.

scholarly journals Essential carboxy groups in xylanase A

1990 ◽  
Vol 270 (1) ◽  
pp. 91-96 ◽  
Author(s):  
M R Bray ◽  
A J Clarke
Keyword(s):  
Catalytic Mechanism ◽  
Ph Dependence ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Water Soluble ◽  
Significant Activity ◽  
Nitrobenzoic Acid ◽  
Woodward’S Reagent K ◽  
Activity Loss

An endo-1,4-beta-xylanase of Schizophyllum commune was purified to homogeneity through a modified procedure employing DEAE-Sepharose CL-6B and gel-filtration chromatography on Sephadex G-50. The role of carboxy groups in the catalytic mechanism was delineated through chemical modification studies. The water-soluble carbodi-imide 1-(4-azonia-4,4-dimethylpentyl)-3-ethylcarbodi-imide iodide (EAC) inactivated the xylanase rapidly and completely in a pseudo-first-order process. Other carbodi-imides and Woodward's Reagent K were less effective in decreasing enzymic activity. Significant protection of the enzyme against EAC inactivation was provided by a mixture of neutral xylo-oligomers. The pH-dependence of the EAC inactivation revealed the presence of a critical ionizable group with a pKa value of 6.6 in the active site of the xylanase. Treatment of the enzyme with diethyl pyrocarbonate resulted in modification of all three histidine residues in the enzyme with 100% retention of original enzymic activity. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetimide and p-chloromercuribenzoate indicated the absence of free/reactive thiol groups. Reaction of the xylanase with tetranitromethane did not result in a significant activity loss as a result of modification of tyrosine residues.

Comparison of the structure and function of ribulosebisphosphate carboxylase–oxygenase from a cold-hardy and nonhardy potato species

1981 ◽  
Vol 59 (4) ◽  
pp. 280-289 ◽  
Author(s):  
Norman P. A. Huner ◽  
Jiwan P. Palta ◽  
Paul H. Li ◽  
John V. Carter
Keyword(s):  
Large Subunit ◽  
Structure And Function ◽  
Sulfhydryl Groups ◽  
Relative Mass ◽  
Nitrobenzoic Acid ◽  
Significant Difference ◽  
Ribulosebisphosphate Carboxylase ◽  
Cold Hardy ◽  
And Function ◽  
Kinetics Of

A comparison of ribulosebisphosphate carboxylase–oxygenase from the leaves of the non-acclimated, cold-hardy species, Solanum commersonii, and the nonacclimated, nonhardy species, Solanum tuberosum showed that this enzyme from the two species differed in structure and function. The results of sulfhydryl group titration with 5,5′-dithiobis(2-nitrobenzoic acid) indicated that the kinetics of titration and the number of accessible sulfhydryl groups in the native enzymes were different. After 30 min, the enzyme from the hardy species had 1.7 times fewer sulfhydryl groups titrated than that from the nonhardy species. In the presence of 1% (w/v) sodium dodecyl sulfate, the total number of sulfhydryl groups titratable with 5,5′-dithiobis-(2-nitrobenzoic acid) was the same for both species. However, this denaturant had a differential effect on the kinetics of titration with 5,5′-dithiobis(2-nitrobenzoic acid). Both enzymes had a native molecular weight of about 550 000. The quaternary structures of the two enzymes were similar with the presence of large and small subunits of 54 000 and 14 000, respectively. However, there was more polypeptide of 108 000 – 110 000 present in preparations of the enzyme from S. tuberosum than from S. commersonii. This polypeptide is an apparent dimer of the large subunit on a relative mass basis. The large subunit of the enzyme from S. tuberosum was more sensitive to the absence of reducing agent and was more sensitive to freezing and thawing than the large subunit of the enzyme from S. commersonii. Catalytic properties of both enzymes at 5 and 25 °C indicated no significant difference in the [Formula: see text] at either temperature. However, the Vmax at 5 °C for the enzyme from S. commersonii was 35% higher than that of the enzyme from S. tuberosum. In contrast, the Vmax at 25 °C for the enzyme of the hardy species was 250% lower than that of the enzyme from the nonhardy species.

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