Examination of Donor Substrate Conversion in Yeast Transketolase

Fiedler, Erik; Golbik, Ralph; Schneider, Günter; Tittmann, Kai; Neef, H; König, Stephan; Hübner, Gerhard

doi:10.1074/jbc.m007936200

Cited by 46 publications

(36 citation statements)

References 34 publications

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“…Instead, we assessed the effects by direct measurements of the apparent dissociation constant for the substrate analog MAP (K d(MAP) ). The K d(MAP) values for charge-reversed variants E401K and K403E were 129-and 136-fold higher, respectively, than with E1ec ( (26,27).…”

Section: Discussionmentioning

confidence: 99%

A Dynamic Loop at the Active Center of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Modulates Substrate Utilization and Chemical Communication with the E2 Component

Kale

Arjunan

Furey

et al. 2007

Journal of Biological Chemistry

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Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.The Escherichia coli pyruvate dehydrogenase multienzyme complex is comprised of the following three enzyme components: E1ec 2 (24 copies, 99,474 Da), E2ec (24 copies, 65,959 Da), and E3ec (12 copies, 50,554 Da) (1-3). The complex catalyzes the oxidative decarboxylation of pyruvate according to Equation 1 (4),The components catalyze parts of the overall reaction (5-7) as shown in Equations 2-6,The E1ec catalyzes thiamin diphosphate (ThDP)-dependent decarboxylation of pyruvate whose product reductively acetylates the lipoamide group of E2ec (supplemental Scheme I). The crystal structure of E1ec with ThDP bound revealed the absence of defined electron density for regions encompassing residues 1-55, 401-413, and 541-557 (8). The first of these regions, the N-terminal one, was shown to interact with E2ec (9). A BLAST search and structure alignment of E1ec with transketolase suggested that a highly conserved residue His-407 of the 401-413 disordered loop (henceforth the "inner" loop) * This work was supported by National Institutes of Health Grants GM 50380and GM 61791. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

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Section: Discussionmentioning

confidence: 99%

A Dynamic Loop at the Active Center of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Modulates Substrate Utilization and Chemical Communication with the E2 Component

Kale

Arjunan

Furey

et al. 2007

Journal of Biological Chemistry

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“…TK was purified according to the protocol of Wikner et al (17), with modifications as described in ref. 15. Circular Dichroism (CD) Measurements.…”

Section: Methodsmentioning

confidence: 99%

“…1). In the absence of a suitable acceptor substrate, the intermediate decomposes into enzyme-ThDP and glycolaldehyde (15), albeit at a rather low rate. This suggested that it would be possible to establish experimental conditions that would lead to significant steady-state levels of this intermediate in the active site of TK, making its detection by biophysical methods possible.…”

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confidence: 99%

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Fiedler

Thorell

Sandalova

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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141

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“…The DYNAFIT method has been used previously (37,38) to characterize complex inhibitory mechanisms. In the example perhaps most relevant to the mechanism proposed for AceAP1, Stanley et al (39) used DYNAFIT to study the inhibition of vitamin K-dependent ␥-glutamylcarboxylase by various polypeptides important in the blood coagulation cascade.…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Ancylostoma Inhibitors of Coagulation Factor Xa

Harrison

Nerlinger

Bungiro

et al. 2002

Journal of Biological Chemistry

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Bloodfeeding hookworms, which currently infect over a billion people in the developing world, are a leading cause of gastrointestinal hemorrhage and iron deficiency anemia. The major anticoagulant inhibitor of coagulation factor Xa has been identified from the hookworm parasite Ancylostoma ceylanicum using reverse transcription PCR and 3-rapid amplification of cDNA ends. This is the first anticoagulant cloned from a hookworm species for which humans are recognized permissive hosts. Despite ϳ50% amino acid similarity, A. ceylanicum anticoagulant peptide 1 (AceAP1) is both immunologically and mechanistically distinct from AcAP5, its homologue isolated from the dog hookworm Ancylostoma caninum. Studies using plasma clotting times and single stage chromogenic assays of factor Xa activity have demonstrated that the recombinant AceAP1 protein is substantially less potent than AcAP5 and that soluble whole worm protein extracts of adult A. ceylanicum possess less anticoagulant activity than extracts of A. caninum. These values correlate with previously reported differences in bloodfeeding capabilities between these two species of hookworm, suggesting that factor Xa inhibitory activity is predictive of hookworm bloodfeeding capabilities in vivo. These fundamental differences in the mechanism of action and immunoreactivity of the major anticoagulant virulence factors from related Ancylostoma hookworm species may have significant implications for human vaccine development.

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Examination of Donor Substrate Conversion in Yeast Transketolase

Cited by 46 publications

References 34 publications

A Dynamic Loop at the Active Center of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Modulates Substrate Utilization and Chemical Communication with the E2 Component

A Dynamic Loop at the Active Center of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Modulates Substrate Utilization and Chemical Communication with the E2 Component

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Molecular Characterization of Ancylostoma Inhibitors of Coagulation Factor Xa

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