Identification of the catalytic glutamate in the E1 component of human pyruvate dehydrogenase

Fang, Rui; Nixon, P.G.F.; Duggleby, Ronald G.

doi:10.1016/s0014-5793(98)01249-6

Cited by 26 publications

(10 citation statements)

References 36 publications

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“…Before determining the specific activity of the recombinantly expressed subunits of PfPDH E1 (a þ b, co-purified) as well as PfOxoDH E1, their purity was verified by SDS-PAGE (data not shown). The enzyme activity was followed by photospectroscopy at 600 nm using the electron acceptor 2,6-dichloroindophenol sodium salt (DCIP) as described previously 55 . The reaction buffer (50 mM KH 2 PO 4 and 2 mM MgCl 2 , pH 7) contained 200 mM TPP, 80 mM DCIP, 2 mM pyruvate/oxoglutarate and 50 mg enzyme.…”

Section: Methodsmentioning

confidence: 99%

Chemical and genetic validation of thiamine utilization as an antimalarial drug target

et al. 2013

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Thiamine is metabolized into an essential cofactor for several enzymes. Here we show that oxythiamine, a thiamine analog, inhibits proliferation of the malaria parasite Plasmodium falciparum in vitro via a thiamine-related pathway and significantly reduces parasite growth in a mouse malaria model. Overexpression of thiamine pyrophosphokinase (the enzyme that converts thiamine into its active form, thiamine pyrophosphate) hypersensitizes parasites to oxythiamine by up to 1,700-fold, consistent with oxythiamine being a substrate for thiamine pyrophosphokinase and its conversion into an antimetabolite. We show that parasites overexpressing the thiamine pyrophosphate-dependent enzymes oxoglutarate dehydrogenase and pyruvate dehydrogenase are up to 15-fold more resistant to oxythiamine, consistent with the antimetabolite inactivating thiamine pyrophosphate-dependent enzymes. Our studies therefore validate thiamine utilization as an antimalarial drug target and demonstrate that a single antimalarial can simultaneously target several enzymes located within distinct organelles.

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Section: Methodsmentioning

confidence: 99%

Chemical and genetic validation of thiamine utilization as an antimalarial drug target

et al. 2013

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“…ThDP is oriented by hydrogen bonds and van der Waals interactions, including the highly conserved l -glutamate residue, which is Glu85 in M. tuberculosis . This residue is implicated in catalysis as demonstrated by several mutagenesis studies. − Substitution of Glu85 with alanine and with isosteric or isofunctional amino acid residues, e.g., l -glutamine or l -aspartate, respectively, led to a dramatic decrease in the activity of the enzyme in comparison to that of the wild type . Amino acid substitutions of conserved residues located in the immediate proximity of Glu85, His84, and Gln86 also led to a decrease in AHAS activity, suggesting that these residues are involved in the stabilization of the Glu85 side chain, keeping it interacting with N1′ of the ThDP .…”

Section: Ilvb/n Acetolactate (Acetohydroxyacid) Synthasementioning

confidence: 97%

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

2017

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The eight enzymes responsible for the biosynthesis of the three branched-chain amino acids (l-isoleucine, l-leucine, and l-valine) were identified decades ago using classical genetic approaches based on amino acid auxotrophy. This review will highlight the recent progress in the determination of the three-dimensional structures of these enzymes, their chemical mechanisms, and insights into their suitability as targets for the development of antibacterial agents. Given the enormous rise in bacterial drug resistance to every major class of antibacterial compound, there is a clear and present need for the identification of new antibacterial compounds with nonoverlapping targets to currently used antibacterials that target cell wall, protein, mRNA, and DNA synthesis.

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“…This residue has been proposed [7] to initiate the ionization of C2 and it has been shown that mutation of this residue greatly diminishes the exchange of the C2 proton with solvent [12,13]. However, this finding is difficult to reconcile with the observation that mutation of this glutamate in transketolase [14], Z. mobilis PDC [15], yeast PDC [16] and human pyruvate dehydrogenase [17] results in enzymes with a significant residual activity.…”

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confidence: 99%

Site‐directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase

Huang¹,

Chang²,

Nixon³

et al. 2001

European Journal of Biochemistry

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Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme about which there is a large body of structural and functional information. The active site contains several absolutely conserved ionizable groups and all of these appear to be important, as judged by the fact that mutation diminishes or abolishes catalytic activity. Previously we have shown [Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby, R.G. (1997) Eur. J. Biochem. 248, 63-71] that the activity is pH-dependent due to changes in kcat/Km while kcat itself is unaffected by pH. The effect on kcat/Km is determined by a group with a pKa of 6.45; the identity of this group has not been determined, although H113 is a possible candidate. Here we mutate five crucial residues in the active site with ionizable side-chains (D27, E50, H113, H114 and E473) in turn, to residues that are nonionizable or should have a substantially altered pKa. Each protein was purified and characterized kinetically. Unexpectedly, the pH-dependence of kcat/Km is largely unaffected in all mutants, ruling out the possibility that any of these five residues is responsible for the observed pKa of 6.45. We conjecture that the kcat/Km profile reflects the protonation of an alcoholate anion intermediate of the catalytic cycle.

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Identification of the catalytic glutamate in the E1 component of human pyruvate dehydrogenase

Cited by 26 publications

References 36 publications

Chemical and genetic validation of thiamine utilization as an antimalarial drug target

Chemical and genetic validation of thiamine utilization as an antimalarial drug target

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

Site‐directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase

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