Significant catalytic roles for Glu47 and Gln 110 in all four of the C-C bond-making and -breaking steps of the reactions of acetohydroxyacid synthase II.


:Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the first common step in the biosynthesis of branched-chain amino acids, condensation of pyruvate with a second 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme II from Escherichia coli is specific for pyruvate as the first donor substrate but exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. In previous studies relying on steady state and transient kinetics, substrate competition and detailed analysis of the distribution of intermediates in the steady-state, we have identified several residues which confer specificity for the donor and acceptor substrates, respectively. Here, we examine the roles of active site polar residues Glu47, Gln110, Lys159, and His251 for elementary steps of catalysis using similar approaches. While Glu47, the conserved essential glutamate conserved in all ThDP-dependent enzymes whose carboxylate is in H-bonding distance of the ThDP iminopyrimidine N1', is involved as expected in cofactor activation, substrate binding, and product elimination, our studies further suggest a crucial catalytic role for it in the carboligation of the acceptor and the hydroxyethyl-ThDP enamine intermediate. The Glu47-cofactor proton shuttle acts in concert with Gln110 in the carboligation. We suggest that either the transient oxyanion on the acceptor carbonyl is stabilized by H-bonding to the glutamine side chain, or carboligation involves glutamine tautomerization and the elementary reactions of addition and protonation occur in a concerted manner. This is in contrast to the situation in other ThDP enzymes that catalyze a carboligation, such as, e.g., transketolase or benzaldehyde lyase, where histidines act as general acid/base catalysts. Our studies further suggest global catalytic roles for Gln110 and Glu47, which are engaged in all major bond-breaking and bond-making steps. In contrast to earlier suggestions, Lys159 has a minor effect on the kinetics and specificity of AHAS II, far less than does Arg276, previously shown to influence the specificity for a 2-ketoacid as a second substrate. His251 has a large effect on donor substrate binding, but this effect masks any other effects of replacement of His251.






Vyazmensky M,Steinmetz A,Meyer D,Golbik R,Barak Z,Tittmann K,Chipman DM




Has Abstract


2011-04-19 00:00:00












  • CO-induced structural rearrangement of the C cluster in Carboxydothermus hydrogenoformans CO dehydrogenase-evidence from Ni K-edge X-ray absorption spectroscopy.

    abstract::We have examined the C cluster in type II CO dehydrogenase (CODH) from Carboxydothermus hydrogenformans using Ni K-edge X-ray absorption near edge spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy. The enzyme was studied under three conditions: "as-isolated" and after treatment with CO or ...


    pub_type: 杂志文章


    authors: Gu W,Seravalli J,Ragsdale SW,Cramer SP

    更新日期:2004-07-20 00:00:00

  • Photoinactivation and photoaffinity labeling of tryptophan synthase alpha 2 beta 2 complex by the product analogue 6-azido-L-tryptophan.

    abstract::The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods. It binds reversibly in the dark to the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (lambda max = 476 nm). The absorbance of this chromophore...


    pub_type: 杂志文章


    authors: Miles EW,Phillips RS

    更新日期:1985-08-13 00:00:00

  • 8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions.

    abstract::Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting partially folded states of proteins. We have carried out a systematic investigation on the effect of ANS, a charged hydrophobic fluorescent dye, on structural properties o...


    pub_type: 杂志文章


    authors: Ali V,Prakash K,Kulkarni S,Ahmad A,Madhusudan KP,Bhakuni V

    更新日期:1999-10-12 00:00:00

  • HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.

    abstract::Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrie...


    pub_type: 杂志文章


    authors: Trent MS,Worsham LM,Ernst-Fonberg ML

    更新日期:1999-07-06 00:00:00

  • DNA binding of methyl-CpG-binding protein MeCP2 in human MCF7 cells.

    abstract::MeCP2 has been identified as a chromatin-associated protein that recognizes MAR elements as well as methyl-CpGs. To characterize target sequences of MeCP2 in human cells, we employed two complementary methods. First, by use of a preparative chromatin immunoprecipitation protocol, we created from MCF7 cells a library e...


    pub_type: 杂志文章


    authors: Koch C,Strätling WH

    更新日期:2004-05-04 00:00:00

  • Self-assembly of aqueous bilirubin ditaurate, a natural conjugated bile pigment, to contraposing enantiomeric dimers and M(-) and P(+) tetramers and their selective hydrophilic disaggregation by monomers and micelles of bile salts.

    abstract::The solution behavior of bilirubin ditaurate (BDT), the first naturally occurring conjugated bile pigment to be physically and chemically characterized, was assessed in aqueous solution and in monomeric and micellar solutions of common taurine-conjugated bile salts (BS). Analytical ultracentrifugation revealed that BD...


    pub_type: 杂志文章


    authors: Neubrand MW,Carey MC,Laue TM

    更新日期:2015-02-24 00:00:00

  • Effects of exposure to low pH on the lateral mobility of influenza hemagglutinin expressed at the cell surface: correlation between mobility inhibition and inactivation.

    abstract::To investigate the possible role of viral glycoprotein mobility in membrane fusion, fluorescence photobleaching recovery was employed to study the effects of exposure to mildly acidic pH (required to convert many viral fusion proteins to the fusion-active form) on the lateral mobility of influenza hemagglutinin (HA) p...


    pub_type: 杂志文章


    authors: Gutman O,Danieli T,White JM,Henis YI

    更新日期:1993-01-12 00:00:00

  • Direct measurement of nucleation and growth rates in lysozyme folding.

    abstract::A kinetic folding intermediate of hen lysozyme is shown to form in a nucleation/growth type of mechanism. Under native solvent conditions, a nucleated state is formed slowly during refolding (tau = 14 +/- 1 ms at 0 M GdmCl) and is rapidly converted to the folding intermediate (tau = 300 +/- 150 micros at 0 M GdmCl). U...


    pub_type: 杂志文章


    authors: Kiefhaber T,Bachmann A,Wildegger G,Wagner C

    更新日期:1997-04-29 00:00:00

  • Membrane topography of the photosynthetic reaction center polypeptides of Rhodopseudomonas sphaeroides.

    abstract::The topography of the photosynthetic reaction center (RC) polypeptides (H, M, and L) was investigated by proteolysis and radioiodination of membrane vesicles isolated from Rhodopseudomonas sphaeroides. Chromatophores, obtained from French-pressed cell lysates, are closed vesicles' and oriented inside out with respect ...


    pub_type: 杂志文章


    authors: Bachmann RC,Gillies K,Takemoto JY

    更新日期:1981-08-04 00:00:00

  • A deuterium surface coil NMR study of the metabolism of D-methionine in the liver of the anesthetized rat.

    abstract::The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR st...


    pub_type: 杂志文章


    authors: London RE,Gabel SA

    更新日期:1988-10-04 00:00:00

  • NMR studies of the phosphotransfer domain of the histidine kinase CheA from Escherichia coli: assignments, secondary structure, general fold, and backbone dynamics.

    abstract::Multidimensional heteronuclear NMR techniques were applied to study the phosphotransfer domain, residues 1-134, of the histidine kinase CheA, from Escherichia coli, which contains the site of autophosphorylation, His48. Assignments of the backbone amide groups and side chain protons are nearly complete. Our studies sh...


    pub_type: 杂志文章


    authors: Zhou H,Lowry DF,Swanson RV,Simon MI,Dahlquist FW

    更新日期:1995-10-24 00:00:00

  • Structure of the human pancreatic cholesterol esterase gene.

    abstract::The gene for human pancreatic cholesterol esterase consists of 11 exons and 10 introns and is 9.2 kb in length. The last and longest exon (841 nucleotides) is unique to the human gene. Functional amino acids are encoded on separate exons. The leader sequence is encoded by a single exon which carries two additional N-t...


    pub_type: 杂志文章


    authors: Kumar BV,Aleman-Gomez JA,Colwell N,Lopez-Candales A,Bosner MS,Spilburg CA,Lowe M,Lange LG

    更新日期:1992-07-07 00:00:00

  • Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli.

    abstract::The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented. The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner. Comparison to the structures of ...


    pub_type: 杂志文章


    authors: Bamford VA,Angove HC,Seward HE,Thomson AJ,Cole JA,Butt JN,Hemmings AM,Richardson DJ

    更新日期:2002-03-05 00:00:00

  • Structural characterization of the ATP-hydrolyzing portion of the coated vesicle proton pump.

    abstract::The ATP-hydrolyzing portion of the proton pump from clathrin-coated vesicles (isolated from calf brain) was solubilized with three nondenaturing detergents (cholate, octyl glucoside, and Triton X-100). The hydrodynamic properties of the solubilized (Mg2+)-ATPase were then determined by sedimentation analysis in H2O an...


    pub_type: 杂志文章


    authors: Forgac M,Berne M

    更新日期:1986-07-29 00:00:00

  • Conserved amino acid residues of the NuoD segment important for structure and function of Escherichia coli NDH-1 (complex I).

    abstract::The NuoD segment (homologue of mitochondrial 49 kDa subunit) of the proton-translocating NADH:quinone oxidoreductase (complex I/NDH-1) from Escherichia coli is in the hydrophilic domain and bears many highly conserved amino acid residues. The three-dimensional structural model of NDH-1 suggests that the NuoD segment, ...


    pub_type: 杂志文章


    authors: Sinha PK,Castro-Guerrero N,Patki G,Sato M,Torres-Bacete J,Sinha S,Miyoshi H,Matsuno-Yagi A,Yagi T

    更新日期:2015-01-27 00:00:00

  • Fourier transform Raman spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: formation of a stable O-like species during light adaptation and detection of its transient N-like photoproduct.

    abstract::Near-infrared FT-Raman spectroscopy can be used to measure the vibrations of the bacteriorhodopsin (bR) chromophore without the disadvantage of conventional visible resonance Raman spectroscopy, where the visible excitation drives the bR photoreactions. We utilized this technique to investigate the light-dark adaptati...


    pub_type: 杂志文章


    authors: Rath P,Krebs MP,He Y,Khorana HG,Rothschild KJ

    更新日期:1993-03-09 00:00:00

  • We FRET so You Don't Have To: New Models of the Lipoprotein Lipase Dimer.

    abstract::Lipoprotein lipase (LPL) is a dimeric enzyme that is responsible for clearing triglyceride-rich lipoproteins from the blood. Although LPL plays a key role in cardiovascular health, an experimentally derived three-dimensional structure has not been determined. Such a structure would aid in understanding mutations in LP...


    pub_type: 杂志文章


    authors: Hayne CK,Yumerefendi H,Cao L,Gauer JW,Lafferty MJ,Kuhlman B,Erie DA,Neher SB

    更新日期:2018-01-16 00:00:00

  • Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics.

    abstract::Thirty single cysteine substitution mutants of T4 lysozyme have been prepared and spin-labeled with a sulfhydryl-specific nitroxide reagent in order to systematically investigate the relationship between nitroxide side-chain mobility and protein structure. The perturbation caused by replacement of a native residue wit...


    pub_type: 杂志文章


    authors: Mchaourab HS,Lietzow MA,Hideg K,Hubbell WL

    更新日期:1996-06-18 00:00:00

  • Partitioning of synaptotagmin I C2 domains between liquid-ordered and liquid-disordered inner leaflet lipid phases.

    abstract::Synaptotagmin I is the calcium sensor in synchronous neurotransmitter release caused by fusion of synaptic vesicles with the presynaptic membrane in neurons. Synaptotagmin I interacts with acidic phospholipids, but also with soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs), at various stages in ...


    pub_type: 杂志文章


    authors: Wan C,Kiessling V,Cafiso DS,Tamm LK

    更新日期:2011-04-05 00:00:00

  • Molten globule formation in apomyoglobin monitored by the fluorescent probe Nile Red.

    abstract::The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (...


    pub_type: 杂志文章


    authors: Polverini E,Cugini G,Annoni F,Abbruzzetti S,Viappiani C,Gensch T

    更新日期:2006-04-25 00:00:00

  • Acid-base equilibrium of the Schiff base in bacteriorhodopsin.

    abstract::Aqueous suspensions of dark-adapted bacteriorhodopsin (bR560) in the purple membrane of Halobacterium halobium are exposed to rapid jumps to high pH. Optical and resonance Raman measurements are carried out by using flow and stationary methods. Above pH congruent to 11.5 bR560 starts to be reversibly converted to a sp...


    pub_type: 杂志文章


    authors: Druckmann S,Ottolenghi M,Pande A,Pande J,Callender RH

    更新日期:1982-09-28 00:00:00

  • Functional groups at the catalytic site of BF1 adenosinetriphosphatase from Escherichia coli.

    abstract::The rates of inactivation of BF1 adenosinetriphosphatase (BF1-ATPase) from Escherichia coli by 7-chloro-4-nitro-2,1,3-benzoxadiazole, 1-fluoro-2,4-dinitrobenzene, 2,4,6-trinitrobenzenesulfonate, phenylglyoxal, and N,N'-dicyclohexylcarbodiimide have been measured in the presence and absence of various concentrations of...


    pub_type: 杂志文章


    authors: Ting LP,Wang JH

    更新日期:1982-01-19 00:00:00

  • Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd.

    abstract::Pf1, a class II filamentous virus, has been investigated by ultraviolet resonance Raman (UVRR) spectroscopy with excitation wavelengths of 257, 244, 238, and 229 nm. The 257-nm UVRR spectrum is rich in Raman bands of the packaged single-stranded DNA (ssDNA) genome, despite the low DNA mass (6%) of the virion. Converse...


    pub_type: 杂志文章


    authors: Wen ZQ,Armstrong A,Thomas GJ Jr

    更新日期:1999-03-09 00:00:00

  • Light-induced conformational changes in photosynthetic reaction centers: impact of detergents and lipids on the electronic structure of the primary electron donor.

    abstract::Light-induced hypsochromic shifts of the Q(y) absorption band of the bacteriochlorophyll dimer (P) from 865 to 850 nm were identified using continuous illumination of dark-adapted reaction centers (RCs) from Rhodobacter capsulatus when dispersed in the most commonly used detergent, the zwitterionic lauryl N-dimethylam...


    pub_type: 杂志文章


    authors: Deshmukh SS,Akhavein H,Williams JC,Allen JP,Kalman L

    更新日期:2011-06-14 00:00:00

  • Identification from a phage display library of peptides that bind to toxic shock syndrome toxin-1 and that inhibit its binding to major histocompatibility complex (MHC) class II molecules.

    abstract::Phage display technique is a powerful tool with which to identify novel binding sequences for antibody and receptor targets. Few studies, however, have used this technology to select affinity peptides for ligand molecules. Here, we screened a peptide phage library for binding to toxic shock syndrome toxin 1 (TSST-1) t...


    pub_type: 杂志文章


    authors: Sato A,Ida N,Fukuyama M,Miwa K,Kazami J,Nakamura H

    更新日期:1996-08-13 00:00:00

  • Thioredoxin reductase-thioredoxin fusion enzyme from Mycobacterium leprae: comparison with the separately expressed thioredoxin reductase.

    abstract::Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. A unique gene organization of TrxR and Trx has been found in Mycobacterium leprae, where TrxR and Trx are encoded by a single gene and, therefore, are expressed as a fusion protein (MlTrxR-Trx). This fusion enzyme is able to catalyze t...


    pub_type: 杂志文章


    authors: Wang PF,Marcinkeviciene J,Williams CH Jr,Blanchard JS

    更新日期:1998-11-17 00:00:00

  • Ionic and nucleotide requirements for microtubule polymerization in vitro.

    abstract::The ionic and nucleotide requirements for the in vitro polymerization of microtubules from purified brain tubulin have been characterized by viscometry. Protein was purified by successive cycles of a temperature dependent assembly-diassembly scheme. Maximal polymerization occurred at a concentration of 0.1 M Pipes (pi...


    pub_type: 杂志文章


    authors: Olmsted JB,Borisy GG

    更新日期:1975-07-01 00:00:00

  • Crystal structure of ribonuclease T1 carboxymethylated at Glu58 in complex with 2'-GMP.

    abstract::The carboxymethylation of RNase T1 at the gamma-carboxyl group of Glu58 leads to a complete loss of the enzymatic activity while it retains substrate-binding ability. Accompanying the carboxymethylation, RNase T1 undergoes a remarkable thermal stabilization of 9 degrees C in the melting temperature (Tm). In order to c...


    pub_type: 杂志文章


    authors: Ishikawa K,Suzuki E,Tanokura M,Takahashi K

    更新日期:1996-06-25 00:00:00

  • Shape and volume of fragments Fab' and (Fab')2 of anti-poly(D-alanyl) antibodies in the presence and absence of tetra-D-alanine as determined by small-angle x-ray scattering.

    abstract::The conformation of two fragments derived from anti-poly(D-alanyl) antibodies, the divalent fragment (Fab')2 and the monovalent fragment Fab', was studied by small-angle X-ray scattering before and after interaction with the tetra-D-alanine amide hapten. More than 90% of the combining sites were occupied by the hapten...


    pub_type: 杂志文章


    authors: Pilz I,Kratky O,Licht A,Sela M

    更新日期:1975-03-25 00:00:00

  • Zinc interactions with regulatory dimers from Escherichia coli aspartate transcarbamoylase.

    abstract::Zn2+ is tetrahedrally bonded to the 4 nonadjacent thiols of each regulatory chain (Mr 17,000) near r-c contacts between catalytic (c) and regulatory chains (r) in aspartate transcarbamoylase (ATCase; c6r6). This paper reports on Zn2+ interactions with r dimer in the absence of stabilizing r-c contacts. After r2 and c3...


    pub_type: 杂志文章


    authors: Jefferson JR,Hunt JB,Ginsburg A

    更新日期:1990-07-17 00:00:00