Cd2+ activation of l-threonine dehydrogenase from Escherichia coli K-12

Craig, P.; Dekker, Eugene E.

doi:10.1016/0167-4838(88)90276-2

Cited by 12 publications

(8 citation statements)

References 14 publications

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“…Similar stimulation (up to 10-fold) by Cd 2＋ was also reported with Escherichia coli threonine dehydrogenase, 43) which is supposed to have a zinc-binding site in which a cysteine residue is located. 44) In addition, Cd 2＋ is known to bind to the sulfhydryl groups of several proteins.…”

Section: Ešect Of Metal Ions On G6pdh Activitysupporting

confidence: 73%

Purification and Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) fromChlorella vulgarisC-27

Honjoh¹,

Mimura²,

Kuroiwa³

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.

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Section: Ešect Of Metal Ions On G6pdh Activitysupporting

confidence: 73%

Purification and Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) fromChlorella vulgarisC-27

Honjoh¹,

Mimura²,

Kuroiwa³

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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“…L-Threonine dehydrogenase was purified from extracts of E. coli K-12 SBD76 cells harboring plasmid pDR121 (Ravnikar & Somerville, 1987) by procedures reported before (Craig & Dekker, 1986) and all samples used were judged to be >99% pure by SDS-PAGE (Weber et al, 1972). The level of protein in enzyme preparations was determined either spectrophotometrically (A280 = 1.106 for 1 mg TDH/mL) (Craig & Dekker, 1986) or by the bicinchoninic acid assay using bovine serum albumin as a standard.…”

Section: Methodsmentioning

confidence: 99%

“…The level of protein in enzyme preparations was determined either spectrophotometrically (A280 = 1.106 for 1 mg TDH/mL) (Craig & Dekker, 1986) or by the bicinchoninic acid assay using bovine serum albumin as a standard. TDH activity was measured spectrophotometrically at 37 "C, pH 8.4, by monitoring the formation of NADH at 340 nm as previously described (Boylan & Dekker, 1981).…”

Section: Methodsmentioning

confidence: 99%

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Woodward's reagent K inactivation of Escherichia coli L‐threonine dehydrogenase: Increased absorbance at 340–350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups

Johnson

Dekker

1996

Protein Science

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L‐Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N‐ethyl‐5‐phenylisoxazolium‐3′‐sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L‐threonine (60 mM) is ∼50% as effective. Tryptic digestion of WRK‐modified TDH followed by HPLC fractionation (pH 6.2) yields four 340‐nm‐absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35–43), whereas peptide II is TAICGTDVHIY (residues 35–45). Peptides not protected are TMLDTMNHGGR (III, residues 248–258) and NCRGGRTHLCR (IV, residues 98–108). Absorbance spectra of these WRK‐peptides were compared with WRK adducts of imidazole, 2‐hydroxy‐ethanethiolate, and acetate. Peptides III and IV have pH‐dependent λmax values (340–350 nm), consistent with histidine modification. Peptide I has a pH‐independent λmax (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys‐38 in the active site of TDH; modification of His‐105 and His‐255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340–350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.

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“…Threonine dehydrogenase has been purified to homogeneity from extracts of chicken liver [5], goat liver [10] and E. coli [11]. Whereas the enzyme from chicken liver is a single polypeptide chain and is inhibited 50% by 1.0 mM Mn 2÷, E. coli threonine dehydrogenase is a tetramer manifesting a basal level of activity in the absence of added metal ions that is stimulated approximately 10-fold by either Mn 2÷ or Cd 2+ [12,13].…”

Section: Introductionmentioning

confidence: 99%

The sulfhydryl content of l-threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation

Craig

Dekker

1990

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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When oxidized to cysteic acid by performic acid or converted to carboxymethylcysteine by alkylation of the reduced enzyme with iodoacetate, a total of six half-cystine residues/subunit are found in L-threonine dehydrogenase (L-threonine: NAD+ oxidoreductase, EC 1.1.1.103; L-threonine + NAD(+)----2-amino-3-oxobutyrate + NADH) from Escherichia coli K-12. Of this total, two exist in disulfide linkage, whereas four are titratable under denaturing conditions by dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), or p-mercuribenzoate. The kinetics of enzyme inactivation and of modification by the latter two reagents indicate that threonine dehydrogenase has no free thiols that selectively react with bulky compounds. While incubation of the enzyme with a large excess of iodoacetamide causes less than 10% loss of activity, the native dehydrogenase is uniquely reactive with and completely inactivated by iodoacetate. The rate of carboxymethylation by iodoacetate of one -SH group/subunit is identical with the rate of inactivation and the carboxymethylated enzyme is no longer able to bind Mn2+. NADH (0.5 mM) provides 40% protection against this inactivation; 60 to 70% protection is seen in the presence of saturating levels of NADH plus L-threonine. Such results coupled with an analysis of the kinetics of inactivation caused by iodoacetate are interpreted as indicating the inhibitor first forms a reversible complex with a positively charged moiety in or near the microenvironment of a reactive -SH group in the enzyme before irreversible alkylation occurs. Specific alkylation of one -SH group/enzyme subunit apparently causes protein conformational changes that entail a loss of catalytic activity and the ability to bind Mn2+.

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Cd2+ activation of l-threonine dehydrogenase from Escherichia coli K-12

Cited by 12 publications

References 14 publications

Purification and Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) fromChlorella vulgarisC-27

Purification and Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) fromChlorella vulgarisC-27

Woodward's reagent K inactivation of Escherichia coli L‐threonine dehydrogenase: Increased absorbance at 340–350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups

The sulfhydryl content of l-threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation

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