Regulation by lipids of cofactor binding to a peripheral membrane enzyme: binding of thiamin pyrophosphate to pyruvate oxidase

O'Brien, Thomas A.; Blake, Robert L.; Gennis, Robert B.

doi:10.1021/bi00633a010

Cited by 32 publications

(10 citation statements)

References 18 publications

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“…However, the trans isomers bind no more strongly than does TPP, which proves that steric factors are important. It has been suggested that the high temperature dependence of the binding constant for TPP to E. coli pyruvate dehydrogenase complex (Moe & Hammes, 1974) and pyruvate oxidase (O'Brien et al, 1977) and of the rate of binding of thiamin thiazolone pyrophosphate to pyruvate oxidase (O'Brien & Gennis, 1980) is further ev-idence for the existence of a hydrophobic interaction in binding TPP. A similar temperature dependence governing the binding of tetrahydro-TPP to E. coli pyruvate dehydrogenase complex was observed (Figure 3).…”

Section: Discussionmentioning

confidence: 99%

Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Lowe¹,

Leeper²,

Perham³

1983

Biochemistry

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Tetrahydrothiamin pyrophosphate, an analogue of thiamin pyrophosphate (TPP) in which the thiazolium ring has been reduced to a thiazolidine ring, was prepared by borohydride reduction of TPP. It consists of four stereoisomers, comprising two diastereomers each of which is a racemic mixture, generated by the introduction of two chiral centers on the thiazolidine ring. The major and minor diastereomers were separated and inferred to be of the cis and trans configurations, respectively, from a study of the nuclear Overhauser effects in the 1H NMR spectrum of the simpler tetrahydrothiamin. Tetrahydro-TPP behaves as a mixture of potent inhibitors of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex from Escherichia coli. The site of binding is probably the TPP-binding site on E1, and the Kd for each of the four stereoisomers was estimated. The cis isomers of tetrahydro-TPP bind more tightly than does TPP, whereas the trans isomers appear to bind with about the same Kd as TPP. Sodium borohydride caused a rapid inhibition of E1 activity in the presence of TPP, believed to be due to reaction of borohydride with enzyme-bound TPP. The experiments are consistent with an enhancement of the reactivity of the thiazole ring of TPP when bound to the catalytic site of E1, which could be due to polarization of the greater than +N=C bond near a hydrophobic or positively charged region of the protein. A spontaneous reactivation occurred after the initial inhibition by borohydride, attributable to a weakly binding inhibitor, not tetrahydro-TPP, being formed at the catalytic site.

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Section: Discussionmentioning

confidence: 99%

Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Lowe¹,

Leeper²,

Perham³

1983

Biochemistry

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“…The lipid-protein interactions, primary hydrophobic (30), have been the subject of intensive studies (for a review, see reference 320). Lipids modulate the turnover number of the enzyme as well as the strength and cooperativity of substrate and cofactor binding (349). Similarly and significantly, the catalytic ligands have a strong influence on the affinity of the protein for lipids (97,431,438).…”

Section: Pyruvate Oxidasementioning

confidence: 99%

The respiratory chains of Escherichia coli.

Ingledew¹,

Poole²

1984

Microbiological Reviews

286

153

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“…The finding that the Triton detergents were unique in binding to the poxB4 oxidase without activating the enzyme suggests that these detergents may interact with the oxidase in an unusual manner. Indeed, previous workers have shown that Triton detergents give the highest level of activation of any lipid activator (1) and that only the Tritons, among the activators tested, increased the dissociation constant of the activated enzyme for TPP-Mg2+ (25).…”

Section: Resultsmentioning

confidence: 94%

Molecular cloning, DNA sequencing, and enzymatic analyses of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids

Chang¹,

Cronan²

1986

J Bacteriol

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Two Escherichia coli pyruvate oxidase (EC 1.2.2.2) mutant genes, poxB3 and poxB4, were cloned on plasmid pBR322. The poxB3 mutant oxidase which was described previously (Y. Y. Chang and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 81: [4348][4349][4350][4351][4352] 1984) was deficient in lipid activation but retained full catalytic activity.The poxB3 mutation was located in the C-terminal half of the gene, and the nucleotide alteration has been determined by DNA sequencing of this part of the gene and by comparing the sequence with that of the wild-type strain (C. Grabau and J. E. Cronan, Jr., submitted for publication). The poxB3 oxidase mutation is the substitution of a serine residue for Pro-536. poxB4, another pyruvate oxidase mutant gene, was also deficient in lipid activation. The major difference between the poxB3 and poxB4 oxidase was in the binding of Triton detergents. The poxB4 mutation was also located in the C-terniinal half of the gene, and sequence analysis has shown that only one nucleotide base was altered, which resulted in Ala-467 being converted to a threonine residue. The results of the amino acid substitutions in the mutant proteins, leading to the functional alteration of the enzyme, are discussed. Preparation of plasmids, DNA sequencing, and other methods involving DNA. Purified plasmids were isolated and prepared by the mild Triton X-100-lysozyme lysis of chloramphenicol-amplified cultures (22) followed by two cycles of cesium chloride-ethidium bromide gradient centrifugation (21). For strains which are lysed poorly by this method (e.g., CG3), the large-scale alkaline lysis method (21) was used.

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Regulation by lipids of cofactor binding to a peripheral membrane enzyme: binding of thiamin pyrophosphate to pyruvate oxidase

Cited by 32 publications

References 18 publications

Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

The respiratory chains of Escherichia coli.

Molecular cloning, DNA sequencing, and enzymatic analyses of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids

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