Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

Stepp, Larry R.; Reed, Lester J.

doi:10.1021/bi00346a026

Cited by 42 publications

(14 citation statements)

References 23 publications

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“…1 is the requirement for TPP as a cofactor, it is tempting to speculate that the conserved sequence motif may be part at least of a common TPP-binding site. Its occurrence in the Ela subunits of the 2-oxo acid dehydrogenase complexes is consistent with this possibility, since the binding site for TPP is thought to reside in the Ela and not the EI~ subunits of the ox kidney and heart pyruvate dehydrogenase complexes [19].…”

Section: A Possible Role For the Sequence Motifsupporting

confidence: 56%

A common structural motif in thiamin pyrophosphate‐binding enzymes

1989

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The amino acid sequences of a wide range of enzymes that utilize thiamin pyrophosphate (TPP) as cofactor have been compared. A common sequence motif approximately 30 residues in length was detected, beginning with the highly conserved sequence -GDG-and concluding with the highly conserved sequence -NN-. Secondary structure predictions suggest that the motif may adopt a flail fold. The same motif was recognised in the primary structure of a protein deduced from the DNA sequence of a hitherto unassigned open reading frame of Rhodobacter capsulata. This putative protein exhibits additional homology with some but not all of the TPP-binding enzymes.

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Section: A Possible Role For the Sequence Motifsupporting

confidence: 56%

A common structural motif in thiamin pyrophosphate‐binding enzymes

1989

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“…2). The reversible character of the inhibition by pyridoxal 5'-phosphate alone, resulting from Schiff base formation with an active-site lysine residue, was corroborated by a characteristic absorption at 340 nm of the pyridoxal-5'phosphate/KCN-treated enzyme, a typical value for a phosphopyridoxyl aminonitrile (Bower and Zalkin, 1982;Stepp and Reed, 1985). The calculated absorption coefficient of 2000 M-' .cm-' is below the absorption coefficient of 10000 M-' .…”

Section: Discussionmentioning

confidence: 94%

“…In this case the product stability will determine whether the adducts can be isolated. Cyanide addition to the azomethine linkage (Scheme 1, pathway 1) forms an aminonitrile resulting in enzyme inactivation, as observed in several cases (Hansen et al, 1974;Bower and Zalkin, 1982;Stepp and Reed, 1985).…”

mentioning

confidence: 79%

Possible involvement of Lys603 from Escherichia coli glucosamine‐6‐phosphate synthase in the binding of its substrate fructose 6‐phosphate

Golinelli‐Pimpaneau

Badet

1991

European Journal of Biochemistry

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Pyridoxal 5′‐phosphate is a competitive inhibitor of glucosamine‐6‐phosphate synthase with respect to the substrate fructose 6‐phosphate. Irreversible inactivation of pyridoxal‐5′‐phosphate‐treated enzyme with [14C]‐cyanide resulted in covalent incorporation of close to 1 mol pyridoxal 5′‐phosphate/mol enzyme subunit. The enzyme‐ pyridoxal‐5′‐phosphate complex could also be inactivated by reduction with NaBH3CN. Sequence analysis of the unique radioactively labelled tryptic peptide, resulting from inactivation with [3H]NaBH3CN, identified the C‐terminal nonapeptide encompassing the modified Lys603. The presence of fructose 6‐phosphate protected this residue from pyridoxylation. Direct evidence that a lysine residue is involved in the binding of the substrate as a Schiff base came from the isolation at 4°C of a enzyme‐fructose‐6‐phosphate complex in a 1:1 molar ratio. Treatment of the enzyme‐[14C]fructose‐6‐phosphate complex with NaBH3CN revealed one site of modification in the tryptic peptide map. In contrast, trapping the same complex with potassium cyanide resulted in the isolation of several radiolabelled peptides containing lysines which could potentially bind fructose 6‐phosphate. However, since the radioactivity was not specifically associated with the lysine residues, it is suggested that these 14C‐labelled peptides resulted from the decomposition of an unstable α,α′‐dihydroxyaminonitrile adduct rather than from a lack of specificity of fructose 6‐phosphate fixation. Lys603 is then the candidate of choice for fructose 6‐phosphate binding since it lies at or near the active site as demonstrated by the trapping experiments with pyridoxal 5′‐phosphate described above, and among the lysines which belong to the sugar‐binding domain this is the only one conserved between the three members of the purF, glutamine‐dependent, amidotransferase subfamily which include the glucosamine‐6‐phosphate synthase from Escherichia coli, Saccharomyces cerevisiae and the Rhizobium nodulation protein NodM.

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“…In PDHCs, the binding site for TPP is thought to reside in the E1α and not the E1β subunits (Stepp & Reed, 1985) and it is in the E1α enzyme that the conserved motif is found. The proposed E1α protein of H. volcanii contains this motif, the level of identity (Fig.…”

Section: The E1α and E1β Componentsmentioning

confidence: 99%

2-Oxoacid dehydrogenase multienzyme complexes in the halophilic Archaea? Gene sequences and protein structural predictions The GenBank accession number for the sequence reported in this paper is AF068743.

et al. 2000

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All Archaea catalyse the conversion of pyruvate to acetyl-CoA via a simple pyruvate oxidoreductase. This is in contrast to the Eukarya and most aerobic bacteria, which use the pyruvate dehydrogenase multienzyme complex [PDHC], consisting of multiple copies of three component enzymes : E1 (pyruvate decarboxylase), E2 (lipoate acetyl-transferase) and E3 (dihydrolipoamide dehydrogenase, DHLipDH). Until now no PDHC activity has been found in the Archaea, although DHLipDH has been discovered in the extremely halophilic Archaea and its gene sequence has been determined. In this paper, the discovery and sequencing of an operon containing the DHLipDH gene in the halophilic archaeon Haloferax volcanii are reported. Upstream of the DHLipDH gene are 3 ORFs which show highest sequence identities with the E1α, E1β and E2 genes of the PDHC from Gram-positive organisms. Structural predictions of the proposed protein product of the E2 gene show a domain structure characteristic of the E2 component in PDHCs, and catalytically important residues, including the lysine to which the lipoic acid cofactor is covalently bound, are conserved. Northern analyses indicate the transcription of the whole operon, but no PDHC enzymic activity could be detected in cell extracts. The presence in the E2 gene of an insertion (equivalent to approximately 100 aa) not found in bacterial or eukaryal E2 proteins, might be predicted to prevent multienzyme complex assembly. This is the first detailed report of the genes for a putative 2-oxoacid dehydrogenase complex in the Archaea, and the evolutionary and metabolic consequences of these findings are discussed.

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Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

Cited by 42 publications

References 23 publications

A common structural motif in thiamin pyrophosphate‐binding enzymes

A common structural motif in thiamin pyrophosphate‐binding enzymes

Possible involvement of Lys603 from Escherichia coli glucosamine‐6‐phosphate synthase in the binding of its substrate fructose 6‐phosphate

2-Oxoacid dehydrogenase multienzyme complexes in the halophilic Archaea? Gene sequences and protein structural predictions The GenBank accession number for the sequence reported in this paper is AF068743.

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