Similar effects by valine and isoleucine on threonine deaminase

Harding, W. M.; Tubbs, J. A.; McDaniel, Deborah

doi:10.1139/o70-126

Cited by 8 publications

(4 citation statements)

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“…In this paper it is reported, for the first time, that native horse muscle acylphosphatase is constituted by two chains SS bound; one, whose C-terminal amino acid is tyrosine and N-terminal histidine, the other, with low molecular weight, which is glutathione. Protein-GSHand protein-cysteine-mixed disulfide have been identified in human plasma albumin (King, 1961), in protein of Ehrlich ascites cells (Revesz and Modig, 1965;Modig, 1968), in bovine serum albumin (Modig, 1968), in rat spleen, liver, heart, muscle, kidney, Yoshida ascites sarcoma (Jackson et al, 1968), and in normal and cataractous human lenses (Harding, 1970).…”

Section: Resultsmentioning

confidence: 99%

Preliminary studies on horse muscle acylphosphatase structure. Evidence of a mixed disulfide with glutathione

Ramponi¹,

Cappugi²,

Treves³

et al. 1971

Biochemistry

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Section: Resultsmentioning

confidence: 99%

Preliminary studies on horse muscle acylphosphatase structure. Evidence of a mixed disulfide with glutathione

Ramponi¹,

Cappugi²,

Treves³

et al. 1971

Biochemistry

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“…The structural similarity among isoleucine, valine, threonine, and their analogs has long been recognized as a possible source of complexity in the analysis of binding isotherms for threonine deaminase (Changeux, 1962; Freundlich & Umbarger, 1963;Harding et al, 1970;Hatfield, 1971;Umbarger, 1973;Decedue et al, 1975; Hofler & Bums, 1978). Competition among similar ligands for either the active sites, the effector sites, or both would complicate any simple analysis of binding isotherms, thereby impeding attempts to reach a consensus for the regulation of the enzyme (Changeux, 1964;Hatfield, 1971;Hofler & Bums, 1978).…”

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confidence: 99%

An Expanded Two-State Model Accounts for Homotropic Cooperativity in Biosynthetic Threonine Deaminase from Escherichia coli

Eisenstein

Yu²,

Fisher³

et al. 1995

Biochemistry

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The linkage between substrate and regulatory effector binding to separate sites on allosteric enzymes results in shifts in their sigmoidal kinetics to regulate metabolism. Control of branched chain amino acid biosynthesis in Escherichia coli occurs in part through shifts in the sigmoidal dependence of alpha-ketobutyrate production promoted by isoleucine and valine binding to biosynthetic threonine deaminase. The structural similarity of threonine, valine, and isoleucine have given rise to suggestions that there may be competition among different ligands for the same sites on this tetrameric enzyme, resulting in a complex pattern of regulation. In an effort to provide a coherent interpretation of the cooperative association of ligands to the active sites and to the effector sites of threonine deaminase, binding studies using single amino acid variants were undertaken. A previously-isolated, feedback-resistant mutant identified in Salmonella typhimurium, ilvA219, has been cloned and sequenced. The phenotype is attributable to a single amino acid substitution in the regulatory domain of the enzyme in which leucine at position 447 is substituted with phenylalanine. The mutant exhibits hyperbolic saturation curves in both ligand binding and steady-state kinetics. These results, in addition to calorimetric and spectroscopic measurements of isoleucine and valine binding, indicate that the low affinity (T) state is destabilized in the mutant and that it exists predominantly in the high affinity (R) conformation in the absence of ligands, providing an explanation for its resistance to isoleucine. Chemical and spectroscopic analyses of another mutant, in which alanine has replaced an essential lysine at position 62 that forms a Schiff base with pyridoxal phosphate, indicate that the cofactor is complexed to exogenous threonine and is therefore unable to bind additional amino acids at the active sites. Isoleucine and valine binding to this inactive, active site-saturated enzyme revealed that it too was stabilized in the R state, yielding binding constants in excellent agreement with the leucine to phenylalanine mutant. The lysine to alanine mutant was further utilized to demonstrate that both threonine and 2-aminobutyrate bind with stronger affinity to the regulatory sites than to the active sites. A direct consequence of these results is that substrates and analogs have a synergistic effect on the allosteric transition since, in effect, they act as both homotropic and heterotropic effectors.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…3). In the absence of isoleucine the wild-type threonine deaminase shows hyperbolic substrate saturation kinetics (4,12); the nH value from the Hill plot is 1. Addition of isoleucine results in a sigmoidal substrate saturation curve (25); the nH value is 1.6 at an isoleucine concentration of I ' 4.…”

Section: Resultsmentioning

confidence: 99%

Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity

Feldner¹,

Grimminger²

1976

J Bacteriol

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The mutant IP7 of Escherichia coli B requires isoleucine or pyridoxine for growth as a consequence of a mutation in the gene coding for biosynthetic threonine deaminase. The mutation of IP7 was shown to be of the nonsense type by the following data: (1) reversion to isoleucine prototrophy involves the formation of external suppression at a high frequency, as shown by transduction experiments; and (ii) the isoleucine requirement is suppressed by lysogenization with a phage carrying the amber suppressor su-3. Cell extracts of the mutant strain contain a low activity of threonine deaminase. The possibility that this activity is biodegradative was ruled out by kinetic experiments. The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al., 1973; Grimminger et al., 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments. Pyridoxal phosphate enhances the maximum catalytic activity of the mutant enzyme by a factor of five, whereas the wild-type enzyme is not affected. In wild-type and mutant threonine deaminase the ratio of protein subunits and bound pyridoxal phosphate is 2:1. The activation of threonine deaminase from strain IP7 is due to a second coenzyme binding site, as shown by (i) spectrophotometric titration of the enzyme with pyridoxal phosphate and by (ii) measurement the pyridoxal phosphate content of the enzyme after sodium borohydride reduction of the protein. The observation of one pyridoxal phosphate binding site per peptide dimer in the wild-type enzyme and of two binding sites per dimer in the mutant strongly suggests that one of the potential sites in the wild-type enzyme is masked by allosteric effects. The factors responsible for the half-of-the-sites reactivity of the coenzyme sites appear to be nonoperative in the mutant protein.

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Similar effects by valine and isoleucine on threonine deaminase

Cited by 8 publications

References 1 publication

Preliminary studies on horse muscle acylphosphatase structure. Evidence of a mixed disulfide with glutathione

Preliminary studies on horse muscle acylphosphatase structure. Evidence of a mixed disulfide with glutathione

An Expanded Two-State Model Accounts for Homotropic Cooperativity in Biosynthetic Threonine Deaminase from Escherichia coli

Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity

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