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












  • Unexpected findings from target analysis of immunoglobulin E and its receptor.

    abstract::The membrane receptor for immunoglobulin E (IgE) and its ligand, IgE, were irradiated with high-energy electrons. Loss of binding activity was measured for each, and the size of the functional targets was assessed. In both cases, the target size was substantially smaller than the covalent structure of the molecule. Th...


    pub_type: 杂志文章


    authors: Fewtrell C,Kempner E,Poy G,Metzger H

    更新日期:1981-11-10 00:00:00

  • Electron-transferring flavoprotein from pig kidney: flavin analogue studies.

    abstract::Apo-electron-transferring flavoprotein from pig kidney (apo-ETF) has been prepared by an acid ammonium sulfate procedure and reconstituted with FAD analogues to probe the flavin binding site. The 8-position of the bound flavin is accessible to solvent as judged by the reaction of 8-Cl-FAD-ETF with sodium sulfide and t...


    pub_type: 杂志文章


    authors: Gorelick RJ,Thorpe C

    更新日期:1986-11-04 00:00:00

  • Enthalpic and entropic effects of salt and polyol osmolytes on site-specific protein-DNA association: the integrase Tn916-DNA complex.

    abstract::The effect of low molecular-weight compounds on the equilibrium constant K(A) can be used to explore the energetics and molecular mechanism of protein-DNA interactions. Here we use the complex composed of the integrase Tn916 DNA-binding domain and its target DNA duplex to investigate the effects of salt and the nonion...


    pub_type: 杂志文章


    authors: Milev S,Bosshard HR,Jelesarov I

    更新日期:2005-01-11 00:00:00

  • Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein.

    abstract::Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repea...


    pub_type: 杂志文章


    authors: Yang Q,de Beer T,Woods L,Meyer JD,Manning MC,Overduin M,Catalano CE

    更新日期:1999-01-05 00:00:00

  • Conformational study of the potent peptide hormone antagonist [1-penicillamine,2-leucine]oxytocin in aqueous solution.

    abstract::[1-Penicillamine,2-leucine]oxytocin is a conformationally restricted analogue of oxytoxin in which the half-cystine-1 and tyrosine-2 residues of the native hormone are replaced by half-penicillamine (beta, beta-dimethyl-half-cystine) and leucine, respectively. This analogue is a surprisingly potent oxytocin antagonist...


    pub_type: 杂志文章


    authors: Mosberg HI,Hruby VJ,Meraldi JP

    更新日期:1981-05-12 00:00:00

  • Sugars Require Rigid Multivalent Displays for Activation of Mouse Sperm Acrosomal Exocytosis.

    abstract::As a prerequisite to mammalian fertilization, the sperm acrosomal vesicle fuses with the plasma membrane and the acrosome contents are exocytosed. Induction occurs through engagement of the sperm receptors by multiple sugar residues. Multivalent polymers displaying mannose, fucose, or GlcNAc are effective synthetic in...


    pub_type: 杂志文章


    authors: Huang H,Rodolis MT,Bhatia SR,Sampson NS

    更新日期:2017-06-06 00:00:00

  • O2 activation by non-heme diiron proteins: identification of a symmetric mu-1,2-peroxide in a mutant of ribonucleotide reductase.

    abstract::Non-heme diiron clusters occur in a number of enzymes (e.g., ribonucleotide reductase, methane monooxygenase, and Delta9-stearoyl-ACP desaturase) that activate O2 for chemically difficult oxidation reactions. In each case, a kinetically labile peroxo intermediate is believed to form when O2 reacts with the diferrous e...


    pub_type: 杂志文章


    authors: Moënne-Loccoz P,Baldwin J,Ley BA,Loehr TM,Bollinger JM Jr

    更新日期:1998-10-20 00:00:00

  • Chemical synthesis of LNA-2-thiouridine and its influence on stability and selectivity of oligonucleotide binding to RNA.

    abstract::Hybridization to RNA is important for many applications, including antisense therapeutics, RNA interference, and microarray screening. Similar thermodynamic stabilities of A-U and G-U base pairs result in difficulties in selective binding to RNA. Moreover, A-U pairs are weaker than G-C pairs so that binding is sometim...


    pub_type: 杂志文章


    authors: Carlucci M,Kierzek E,Olejnik A,Turner DH,Kierzek R

    更新日期:2009-11-24 00:00:00

  • Effect of base, pentose, and phosphodiester backbone structures on binding and repair of pyrimidine dimers by Escherichia coli DNA photolyase.

    abstract::Photolyases reverse the effects of UV light on cells by converting cyclobutane dipyrimidine photoproducts (pyrimidine dimers, Pyr mean value of Pyr) into pyrimidine monomers in a light-dependent reaction. Previous work has suggested that, based on substrate preference, there are two classes of photolyase: DNA photolya...


    pub_type: 杂志文章


    authors: Kim ST,Sancar A

    更新日期:1991-09-03 00:00:00

  • Structure and action of heteronemertine polypeptide toxins. Membrane penetration by Cerebratulus lacteus toxin A-III.

    abstract::The heteronemertine worm Cerebratulus lacteus produces a family of three structurally homologous proteins that function as direct lytic factors for a variety of cells [Kem, W. R., & Blumenthal, K. M. (1978) J. Biol. Chem. 253, 5752-5757]. It is demonstrated herein that the hemolytic activity of the most abundant varia...


    pub_type: 杂志文章


    authors: Blumenthal KM

    更新日期:1982-08-31 00:00:00

  • Stability of annexin V in ternary complexes with Ca2+ and anionic phospholipids: IR studies of monolayer and bulk phases.

    abstract::Annexin V (AxV) is a member of a family of proteins that exhibit functionally relevant Ca2+-dependent binding to anionic phospholipid membranes. Protein structure and stability as a function of Ca2+ and phospholipids was studied by bulk phase infrared (IR) spectroscopy and by IR reflection-absorption spectroscopy (IRR...


    pub_type: 杂志文章


    authors: Wu F,Flach CR,Seaton BA,Mealy TR,Mendelsohn R

    更新日期:1999-01-12 00:00:00

  • Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin).

    abstract::The mechanism of activation of cGMP phosphodiesterase by the GTP-binding protein in the disc membrane of retinal rods has been investigated by measuring the light-induced phosphodiesterase activity in reconstituted systems where the concentration of either the GTP-binding protein or the phosphodiesterase is varied. Th...


    pub_type: 杂志文章


    authors: Bennett N,Clerc A

    更新日期:1989-09-05 00:00:00

  • Thermodynamic cycle analysis and inhibitor design against beta-lactamase.

    abstract::Beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and cephalosporins. Transition-state analogues that bind to the enzymes with nanomolar affinities have been introduced in an effort to reverse the resistance conferred by these enzymes. To understand the or...


    pub_type: 杂志文章


    authors: Roth TA,Minasov G,Morandi S,Prati F,Shoichet BK

    更新日期:2003-12-16 00:00:00

  • A Novel Mechanism for Calmodulin-Dependent Inactivation of Transient Receptor Potential Vanilloid 6.

    abstract::The paralogues TRPV5 and TRPV6 belong to the vanilloid subfamily of the transient receptor potential (TRP) superfamily of ion channels, and both play an important role in overall Ca2+ homeostasis. The functioning of the channels centers on a tightly controlled Ca2+-dependent feedback mechanism in which the direct bind...


    pub_type: 杂志文章


    authors: Bate N,Caves RE,Skinner SP,Goult BT,Basran J,Mitcheson JS,Vuister GW

    更新日期:2018-05-08 00:00:00

  • Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone.

    abstract::Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hyd...


    pub_type: 杂志文章


    authors: Addya S,Zheng YM,Shayiq RM,Fan JY,Avadhani NG

    更新日期:1991-08-27 00:00:00

  • Circular Dichroism Studies of Cyanate-Induced Conformational Changes in Hemoglobins A and S.

    abstract::Circular dichroism and difference spectroscopy have been used to study dilute aqueous solutions of oxygenated, deoxygenated, and carbamoylated deoxygenated hemoglobins A and S (HbA and HbS, respectively). The spectra of HbA and HbS, in comparable state of oxygenation or carbamoylation, are identical, strongly implying...


    pub_type: 杂志文章


    authors: Simons ER,Hartzband P,Whitin J,Chapman C

    更新日期:1976-09-07 00:00:00

  • 1H NMR dipolar echo decay spectroscopy: a sensitive probe of membrane structure.

    abstract::The structural and motional properties of membrane lipids in various physical states and macroscopic organizations are elucidated by 1H NMR dipolar echo decay spectroscopy (DECODE). Multilamellar lipid dispersions in the gel (L beta') and liquid-crystalline (L alpha) states and a nonbilayer, hexagonal (HII) phase are ...


    pub_type: 杂志文章


    authors: Janes N,Rubin E,Taraschi TF

    更新日期:1990-09-11 00:00:00

  • Thermodynamics and NMR of internal G.T mismatches in DNA.

    abstract::Thermodynamics of 39 oligonucleotides with internal G.T mismatches dissolved in 1 M NaCl were determined from UV absorbance versus temperature profiles. These data were combined with literature values of six sequences to derive parameters for 10 linearly independent trimer and tetramer sequences with G.T mismatches an...


    pub_type: 杂志文章


    authors: Allawi HT,SantaLucia J Jr

    更新日期:1997-08-26 00:00:00

  • Thermodynamics of reversible and irreversible unfolding and domain interactions of glucoamylase from Aspergillus niger studied by differential scanning and isothermal titration calorimetry.

    abstract::The stability of three forms of glucoamylase from Aspergillus niger has been investigated by differential scanning and isothermal titration calorimetry: Glucoamylase 1 (GA1), which consists of a catalytic domain and a starch-binding domain (SBD) connected by a heavily O-glycosylated linker region; glucoamylase 2 (GA2)...


    pub_type: 杂志文章


    authors: Christensen T,Svensson B,Sigurskjold BW

    更新日期:1999-05-11 00:00:00

  • NMR characterization of three-disulfide variants of lysozyme, C64A/C80A, C76A/C94A, and C30A/C115A--a marginally stable state in folded proteins.

    abstract::Our earlier NMR study showed that a two-disulfide variant of hen lysozyme containing intra-alpha-domain disulfide bridges, C6-C127 and C30-C115, is partially folded, with the alpha domain tightly folded to the nativelike conformation and the beta domain flexible or unfolded. With a view that the formation of a third d...


    pub_type: 杂志文章


    authors: Yokota A,Hirai K,Miyauchi H,Iimura S,Noda Y,Inoue K,Akasaka K,Tachibana H,Segawa S

    更新日期:2004-06-01 00:00:00

  • Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii.

    abstract::Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii was inactivated by low concentrations of the lysine-specific reagent pyridoxal phosphate (PLP) after sodium borohydride reduction. The substrates fructose 6-phosphate and fructose 1,6-bisphosphate protected against inactiv...


    pub_type: 杂志文章


    authors: Green PC,Latshaw SP,Ladror US,Kemp RG

    更新日期:1992-05-26 00:00:00

  • Molecular structure and polymorphic map of the human phenylalanine hydroxylase gene.

    abstract::Human phenylalanine hydroxylase is a liver-specific enzyme that catalyzes the conversion of phenylalanine to tyrosine. Absence of enzymatic activity results in phenylketonuria, a genetic disorder that causes development of severe mental retardation in untreated children. In this paper we report the cloning and structu...


    pub_type: 杂志文章


    authors: DiLella AG,Kwok SC,Ledley FD,Marvit J,Woo SL

    更新日期:1986-02-25 00:00:00

  • Crystal structure of recombinant human platelet factor 4.

    abstract::The crystal structure of human platelet factor 4 (PF4) has been solved to a resolution of 2.4 A by molecular replacement and refined to an R-factor of 24.1%. The structure consists of four polypeptide chains which form a tetrameric unit. N-terminal residues, previously defined as a random coil or extended loop region,...


    pub_type: 杂志文章


    authors: Zhang X,Chen L,Bancroft DP,Lai CK,Maione TE

    更新日期:1994-07-12 00:00:00

  • Structural uncoupling between opposing domains of oxidized calmodulin underlies the enhanced binding affinity and inhibition of the plasma membrane Ca-ATPase.

    abstract::Stabilization of the plasma membrane Ca-ATPase (PMCA) in an inactive conformation upon oxidation of multiple methionines in the calcium regulatory protein calmodulin (CaM) is part of an adaptive cellular response to minimize ATP utilization and the generation of reactive oxygen species (ROS) under conditions of oxidat...


    pub_type: 杂志文章


    authors: Chen B,Mayer MU,Squier TC

    更新日期:2005-03-29 00:00:00

  • Concentration of activated intermediates of the fructose-1,6-bisphosphate aldolase and triosephosphate isomerase reactions.

    abstract::As discovered by Grazi & Trombetta [Grazi, E., & Trombetta, G. (1978) Biochem. J. 175, 361], fructose-1,6-bisphosphate aldolase of rabbit muscle causes the slow formation of inorganic phosphate (Pi) and methylglyoxal when incubated with dihydroxyacetone phosphate (DHAP). In addition, these authors found an acid-labile...


    pub_type: 杂志文章


    authors: Iyengar R,Rose IA

    更新日期:1981-03-03 00:00:00

  • Analysis of the ternary interaction of the red cell membrane skeletal proteins spectrin, actin, and 4.1.

    abstract::Spectrin dimers interact weakly with F-actin under physiological solvent conditions (with an association constant of about 5 X 10(3) M-1 at 20 degrees C). In the presence of the membrane skeletal constituent, protein 4.1, strong binding is observed; an analysis of the profiles for formation of a ternary complex leads ...


    pub_type: 杂志文章


    authors: Ohanian V,Wolfe LC,John KM,Pinder JC,Lux SE,Gratzer WB

    更新日期:1984-09-11 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

  • Cationic copper(II) porphyrins intercalate into domains of double-stranded RNA.

    abstract::A cationic, copper(II)-containing ligand, derived from bulky 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, Cu(T4), and two sterically friendlier forms, [trans-5,15-di(N-methylpyridinium-4-yl)porphyrinato]copper(II), Cu(tD4), and [cis-5,10-di(N-methylpyridinium-4-yl)porphyrinato]copper(II), Cu(cD4), bind to DN...


    pub_type: 杂志文章


    authors: Briggs BN,Gaier AJ,Fanwick PE,Dogutan DK,McMillin DR

    更新日期:2012-09-25 00:00:00

  • 9-(5',6'-dideoxy-beta-D-ribo-hex-5'-ynofuranosyl)adenine, a novel irreversible inhibitor of S-adenosylhomocysteine hydrolase.

    abstract::The acetylenic analogue of adenosine 9-(5',6'-dideoxy-beta-D-ribo-hex-5'-ynofuranosyl)adenine has been synthesized, and its behavior as an inhibitor of bovine S-adenosylhomocysteine hydrolase has been examined. Incubation of the enzyme with excess inhibitor caused a time-dependent, irreversible inactivation of the enz...


    pub_type: 杂志文章


    authors: Parry RJ,Muscate A,Askonas LJ

    更新日期:1991-10-15 00:00:00

  • The flavoprotein component of the Escherichia coli sulfite reductase: expression, purification, and spectral and catalytic properties of a monomeric form containing both the flavin adenine dinucleotide and the flavin mononucleotide cofactors.

    abstract::The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain. SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E. coli and purified as a monomer. The N-terminal part of the native protein contains thus al...


    pub_type: 杂志文章


    authors: Zeghouf M,Fontecave M,Macherel D,Covès J

    更新日期:1998-04-28 00:00:00