Participation of cysteinyl residues in the structure and function of muscle aldolase. Characterization of mixed bisulfide derivatives

Howard M. Steinman, Frederic M. Richards

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Cysteinyl residues in rabbit muscle D-fructose 1,6-diphosphate (FDP) aldolase (EC 4.1.2.13) were converted into mixed disulfides by treatment with a series of disulfide monosulfoxides (RCH2CH2S(=O)SCH2CH2R, R = carboxyl, amino, or carbamyl) at pH 7.0-7.4. Regardless of the R group in the reagent, two of the eight cysteinyl residues per subunit were reactive in the presence of the competitive inhibitor, hexitol 1,6-diphosphate (HDP) and two additional residues reactive only in the absence of HDP. The remaining four cysteinyl residues were largely unmodified whether the inhibitor was present or absent. These sulfhydryl groups of aldolase were thus divisible into three classes, exposed, protected, and buried, the classes containing two, two, and four residues per subunit, respectively. The two exposed groups were localized in the two cyanogen bromide peptides derived from the carboxyl end of the subunit polypeptide chain; the two protected groups were located one each in the amino and carboxyl CNBr peptides, and the four buried groups in the two CNBr peptides from the amino end of the subunit chain. Derivatives modified in the presence of HDP exhibited only slight changes in the kinetic parameters for FDP cleavage. Significant decreases (50-80%) in Vm and increases (30- to 100-fold) in Km occurred when the two protected SH groups were subsequently modified, with the changes for the neutral, R = carbamyl, derivative being smaller than for the charged carboxyl and amino derivatives. The denaturation of unmodified aldolase by guanidine hydrochloride (Gd·HCl) at pH 5.5 was shown to be composed of two steps: (1) dissociation of tetramer directly to monomer, accompanied by loss of a significant portion of the ordered secondary structure, and (2) disruption of the residual structure of the dissociated subunits. All mixed disulfide derivatives were similar in exhibiting a two-step mechanism of denaturation, and were practically identical in the Gd·HCl dependence of the second step. They were different from the unmodified protein only in the Gd·HCl sensitivity of the first step, where, for each R, the order of decreasing stability was unmodified aldolase, HDP-protected derivative, then unprotected derivative. Within each class of three protected and three unprotected derivatives, the R = amino proteins were least stable to Gd·HCl, and the carbamyl and carboxyl proteins of comparable stability. At neutral pH, the disulfide cystamine modified one reactive SH group per subunit, identified as that in the CNBr peptide preceding the carboxyl-terminal CNBr peptide, but had no significant deleterious effect on the catalytic or structural properties of the protein.

Original languageEnglish (US)
Pages (from-to)4360-4372
Number of pages13
JournalBiochemistry
Volume9
Issue number22
StatePublished - 1970
Externally publishedYes

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Fructose-Bisphosphate Aldolase
Muscle
Diphosphates
Guanidine
Derivatives
Disulfides
Muscles
Peptides
Denaturation
Cystamine
Proteins
Cyanogen Bromide
Protein Stability
Kinetic parameters
Structural properties
Rabbits
Monomers
hexitol

ASJC Scopus subject areas

  • Biochemistry

Cite this

Participation of cysteinyl residues in the structure and function of muscle aldolase. Characterization of mixed bisulfide derivatives. / Steinman, Howard M.; Richards, Frederic M.

In: Biochemistry, Vol. 9, No. 22, 1970, p. 4360-4372.

Research output: Contribution to journalArticle

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abstract = "Cysteinyl residues in rabbit muscle D-fructose 1,6-diphosphate (FDP) aldolase (EC 4.1.2.13) were converted into mixed disulfides by treatment with a series of disulfide monosulfoxides (RCH2CH2S(=O)SCH2CH2R, R = carboxyl, amino, or carbamyl) at pH 7.0-7.4. Regardless of the R group in the reagent, two of the eight cysteinyl residues per subunit were reactive in the presence of the competitive inhibitor, hexitol 1,6-diphosphate (HDP) and two additional residues reactive only in the absence of HDP. The remaining four cysteinyl residues were largely unmodified whether the inhibitor was present or absent. These sulfhydryl groups of aldolase were thus divisible into three classes, exposed, protected, and buried, the classes containing two, two, and four residues per subunit, respectively. The two exposed groups were localized in the two cyanogen bromide peptides derived from the carboxyl end of the subunit polypeptide chain; the two protected groups were located one each in the amino and carboxyl CNBr peptides, and the four buried groups in the two CNBr peptides from the amino end of the subunit chain. Derivatives modified in the presence of HDP exhibited only slight changes in the kinetic parameters for FDP cleavage. Significant decreases (50-80{\%}) in Vm and increases (30- to 100-fold) in Km occurred when the two protected SH groups were subsequently modified, with the changes for the neutral, R = carbamyl, derivative being smaller than for the charged carboxyl and amino derivatives. The denaturation of unmodified aldolase by guanidine hydrochloride (Gd·HCl) at pH 5.5 was shown to be composed of two steps: (1) dissociation of tetramer directly to monomer, accompanied by loss of a significant portion of the ordered secondary structure, and (2) disruption of the residual structure of the dissociated subunits. All mixed disulfide derivatives were similar in exhibiting a two-step mechanism of denaturation, and were practically identical in the Gd·HCl dependence of the second step. They were different from the unmodified protein only in the Gd·HCl sensitivity of the first step, where, for each R, the order of decreasing stability was unmodified aldolase, HDP-protected derivative, then unprotected derivative. Within each class of three protected and three unprotected derivatives, the R = amino proteins were least stable to Gd·HCl, and the carbamyl and carboxyl proteins of comparable stability. At neutral pH, the disulfide cystamine modified one reactive SH group per subunit, identified as that in the CNBr peptide preceding the carboxyl-terminal CNBr peptide, but had no significant deleterious effect on the catalytic or structural properties of the protein.",
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T1 - Participation of cysteinyl residues in the structure and function of muscle aldolase. Characterization of mixed bisulfide derivatives

AU - Steinman, Howard M.

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N2 - Cysteinyl residues in rabbit muscle D-fructose 1,6-diphosphate (FDP) aldolase (EC 4.1.2.13) were converted into mixed disulfides by treatment with a series of disulfide monosulfoxides (RCH2CH2S(=O)SCH2CH2R, R = carboxyl, amino, or carbamyl) at pH 7.0-7.4. Regardless of the R group in the reagent, two of the eight cysteinyl residues per subunit were reactive in the presence of the competitive inhibitor, hexitol 1,6-diphosphate (HDP) and two additional residues reactive only in the absence of HDP. The remaining four cysteinyl residues were largely unmodified whether the inhibitor was present or absent. These sulfhydryl groups of aldolase were thus divisible into three classes, exposed, protected, and buried, the classes containing two, two, and four residues per subunit, respectively. The two exposed groups were localized in the two cyanogen bromide peptides derived from the carboxyl end of the subunit polypeptide chain; the two protected groups were located one each in the amino and carboxyl CNBr peptides, and the four buried groups in the two CNBr peptides from the amino end of the subunit chain. Derivatives modified in the presence of HDP exhibited only slight changes in the kinetic parameters for FDP cleavage. Significant decreases (50-80%) in Vm and increases (30- to 100-fold) in Km occurred when the two protected SH groups were subsequently modified, with the changes for the neutral, R = carbamyl, derivative being smaller than for the charged carboxyl and amino derivatives. The denaturation of unmodified aldolase by guanidine hydrochloride (Gd·HCl) at pH 5.5 was shown to be composed of two steps: (1) dissociation of tetramer directly to monomer, accompanied by loss of a significant portion of the ordered secondary structure, and (2) disruption of the residual structure of the dissociated subunits. All mixed disulfide derivatives were similar in exhibiting a two-step mechanism of denaturation, and were practically identical in the Gd·HCl dependence of the second step. They were different from the unmodified protein only in the Gd·HCl sensitivity of the first step, where, for each R, the order of decreasing stability was unmodified aldolase, HDP-protected derivative, then unprotected derivative. Within each class of three protected and three unprotected derivatives, the R = amino proteins were least stable to Gd·HCl, and the carbamyl and carboxyl proteins of comparable stability. At neutral pH, the disulfide cystamine modified one reactive SH group per subunit, identified as that in the CNBr peptide preceding the carboxyl-terminal CNBr peptide, but had no significant deleterious effect on the catalytic or structural properties of the protein.

AB - Cysteinyl residues in rabbit muscle D-fructose 1,6-diphosphate (FDP) aldolase (EC 4.1.2.13) were converted into mixed disulfides by treatment with a series of disulfide monosulfoxides (RCH2CH2S(=O)SCH2CH2R, R = carboxyl, amino, or carbamyl) at pH 7.0-7.4. Regardless of the R group in the reagent, two of the eight cysteinyl residues per subunit were reactive in the presence of the competitive inhibitor, hexitol 1,6-diphosphate (HDP) and two additional residues reactive only in the absence of HDP. The remaining four cysteinyl residues were largely unmodified whether the inhibitor was present or absent. These sulfhydryl groups of aldolase were thus divisible into three classes, exposed, protected, and buried, the classes containing two, two, and four residues per subunit, respectively. The two exposed groups were localized in the two cyanogen bromide peptides derived from the carboxyl end of the subunit polypeptide chain; the two protected groups were located one each in the amino and carboxyl CNBr peptides, and the four buried groups in the two CNBr peptides from the amino end of the subunit chain. Derivatives modified in the presence of HDP exhibited only slight changes in the kinetic parameters for FDP cleavage. Significant decreases (50-80%) in Vm and increases (30- to 100-fold) in Km occurred when the two protected SH groups were subsequently modified, with the changes for the neutral, R = carbamyl, derivative being smaller than for the charged carboxyl and amino derivatives. The denaturation of unmodified aldolase by guanidine hydrochloride (Gd·HCl) at pH 5.5 was shown to be composed of two steps: (1) dissociation of tetramer directly to monomer, accompanied by loss of a significant portion of the ordered secondary structure, and (2) disruption of the residual structure of the dissociated subunits. All mixed disulfide derivatives were similar in exhibiting a two-step mechanism of denaturation, and were practically identical in the Gd·HCl dependence of the second step. They were different from the unmodified protein only in the Gd·HCl sensitivity of the first step, where, for each R, the order of decreasing stability was unmodified aldolase, HDP-protected derivative, then unprotected derivative. Within each class of three protected and three unprotected derivatives, the R = amino proteins were least stable to Gd·HCl, and the carbamyl and carboxyl proteins of comparable stability. At neutral pH, the disulfide cystamine modified one reactive SH group per subunit, identified as that in the CNBr peptide preceding the carboxyl-terminal CNBr peptide, but had no significant deleterious effect on the catalytic or structural properties of the protein.

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