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359 a.a.
324 a.a.
42 a.a.
35 a.a.
_MG
__K
 Pyruvate |
+ |
[dihydrolipoyllysine-residue acetyltransferase] lipoyllysine |
= |
[dihydrolipoyllysine-residue acetyltransferase] S- acetyldihydrolipoyllysine |
+ |
 CO(2) |
|---|
Bound ligand (Het Group name = TDP) corresponds exactly |
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 Acetyl-CoA |
+ |
enzyme N(6)-(dihydrolipoyl)lysine |
= |
 CoA |
+ |
enzyme N(6)-(S- acetyldihydrolipoyl)lysine |
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Key reference
DOI no: 10.1126/science.1101030 Science 306:872-876 (2004) PubMed id: 15514159
A molecular switch and proton wire synchronize the active sites in thiamine enzymes. R.A.Frank, C.M.Titman, J.V.Pratap, B.F.Luisi, R.N.Perham. ABSTRACT
Thiamine diphosphate (ThDP) is used as a cofactor in many key metabolic enzymes. We present evidence that the ThDPs in the two active sites of the E1 (EC 1.2.4.1) component of the pyruvate dehydrogenase complex communicate over a distance of 20 angstroms by reversibly shuttling a proton through an acidic tunnel in the protein. This "proton wire" permits the co-factors to serve reciprocally as general acid/base in catalysis and to switch the conformation of crucial active-site peptide loops. This synchronizes the progression of chemical events and can account for the oligomeric organization, conformational asymmetry, and "ping-pong" kinetic properties of E1 and other thiamine-dependent enzymes.
Selected figure(s)
Figure 2.
Fig. 2. B. stearothermophilus E1 active-site asymmetry and proposed proton wire. (A) Ribbon diagram of E1 taken from its complex with the PSBD of E2 colored by temperature (B-) factor. The ThDP cofactors are represented by space-filling atoms and three Mg2+ ions (red spheres), one in each of the two active sites and one midway in the protein tunnel-like cavity that links them. The dyad axis of E1 is oriented vertically. E1 is symmetric except for residues in the active sites, which are non-equivalent. Two peptide loops (275 to 293 and
Figure 4.
Fig. 4. Catalytic mechanism of ThDP-dependent enzymes. (A to D) are the steps of ThDP activation in both active sites and (E) is the slinky cycle. (A) ThDP binds fast/tightly to the first site and is activated to generate a ThDP C2-carbanion. The ThDP C2 proton is relayed via the amino N4' group to the N1' atom of the ThDP aminopyrimidine ring and onward through a proton wire to the open, apo-active site. (B) Two loops (represented by a blue shape) preorganize the active site by folding around the zwitterionic thiazolium. After the first site is activated, the second site has no route for abstracting a second proton, so its ThDP binds but remains dormant. (C) Substrate (in this example, pyruvate) reacts with the activated C2. The ThDP of the second active site is a general acid, donating a proton to the first site. (D) This results in decarboxylation at the first site (not shown, see Fig. 4E), which forms the enamine intermediate, and activation of the second site. Both active sites now gain entry into the slinky cycle, shown in panel (E). The entry points into the slinky cycle are nonequivalent for each active site and are highlighted by an asterisk and a green border. At the first and last steps of ping-pong catalysis, both ThDPs separated by a 20 Å proton wire are mutually obligated as general acid-base catalysts in a slinky-like progression of chemical events. The dithiolane ring of the lipoyl domain is the second substrate in this example and requires activation by ßHis128 in the active site (10, 11).The above figures are reprinted by permission from the AAAs: Science (2004, 306, 872-876) copyright 2004. Figures were selected by the author.
Literature references that cite this PDB file's key reference
PubMed id Reference
19476486 B.Shaanan, and D.M.Chipman (2009).
Reaction mechanisms of thiamin diphosphate enzymes: new insights into the role of a conserved glutamate residue.FEBS J, 276, 2447-2453.
19490097 K.Agyei-Owusu, and F.J.Leeper (2009).
Thiamin diphosphate in biological chemistry: analogues of thiamin diphosphate in studies of enzymes and riboswitches.FEBS J, 276, 2905-2916.
19476485 N.S.Nemeria, S.Chakraborty, A.Balakrishnan, and F.Jordan (2009).
Reaction mechanisms of thiamin diphosphate enzymes: defining states of ionization and tautomerization of the cofactor at individual steps.FEBS J, 276, 2432-3446.
19771336 S.R.Meech (2009).
Excited state reactions in fluorescent proteins.Chem Soc Rev, 38, 2922-2934.
18515642 A.Thomaeus, A.Naworyta, S.L.Mowbray, and M.Widersten (2008).
Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability.Protein Sci, 17, 1275-1284.
PDB code: 3cxu
18725455 I.Mochalkin, J.R.Miller, A.Evdokimov, S.Lightle, C.Yan, C.K.Stover, and G.L.Waldrop (2008).
Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase.Protein Sci, 17, 1706-1718.
PDB codes: 2c00 2j9g 2vpq 2vqd 2vr1
17969139 J.P.Aucamp, R.J.Martinez-Torres, E.G.Hibbert, and P.A.Dalby (2008).
A microplate-based evaluation of complex denaturation pathways: structural stability of Escherichia coli transketolase.Biotechnol Bioeng, 99, 1303-1310.
18058864 M.Alstrup Lie, and B.Schiøtt (2008).
A DFT study of solvation effects on the tautomeric equilibrium and catalytic ylide generation of thiamin models.J Comput Chem, 29, 1037-1047.
18988747 P.Neumann, A.Weidner, A.Pech, M.T.Stubbs, and K.Tittmann (2008).
Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli.Proc Natl Acad Sci U S A, 105, 17390-17395.
PDB codes: 3ey9 3eya 19812737 R.Wittig, and J.F.Coy (2008).
The role of glucose metabolism and glucose-associated signalling in cancer.Perspect Medicin Chem, 1, 64-82.
18004749 V.I.Bunik, and D.Degtyarev (2008).
Structure-function relationships in the 2-oxo acid dehydrogenase family: substrate-specific signatures and functional predictions for the 2-oxoglutarate dehydrogenase-like proteins.Proteins, 71, 874-890.
19081062 X.Y.Pei, C.M.Titman, R.A.Frank, F.J.Leeper, and B.F.Luisi (2008).
Snapshots of catalysis in the E1 subunit of the pyruvate dehydrogenase multienzyme complex.Structure, 16, 1860-1872.
PDB codes: 3duf 3dv0 3dva
17311134 K.M.Erixon, C.L.Dabalos, and F.J.Leeper (2007).
Inhibition of pyruvate decarboxylase from Z. mobilis by novel analogues of thiamine pyrophosphate: investigating pyrophosphate mimics.Chem Commun (Camb), 0, 960-962.
17182735 N.Nemeria, S.Chakraborty, A.Baykal, L.G.Korotchkina, M.S.Patel, and F.Jordan (2007).
The 1',4'-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes.Proc Natl Acad Sci U S A, 104, 78-82.
17403037 W.Versées, S.Spaepen, J.Vanderleyden, and J.Steyaert (2007).
The crystal structure of phenylpyruvate decarboxylase from Azospirillum brasilense at 1.5 A resolution. Implications for its catalytic and regulatory mechanism.FEBS J, 274, 2363-2375.
PDB code: 2nxw
16699828 J.A.McCourt, and R.G.Duggleby (2006).
Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids.Amino Acids, 31, 173-210.
16938834 M.M.Rhodes, K.Réblová, J.Sponer, and N.G.Walter (2006).
Trapped water molecules are essential to structural dynamics and function of a ribozyme.Proc Natl Acad Sci U S A, 103, 13380-13385.
16284263 P.Leiderman, D.Huppert, and N.Agmon (2006).
Transition in the temperature-dependence of GFP fluorescence: from proton wires to proton exit.Biophys J, 90, 1009-1018. The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.
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