Abstract:
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Vyazmensky M,Steinmetz A,Meyer D,Golbik R,Barak Z,Tittmann K,Chipman DMdoi
10.1021/bi102051hsubject
Has Abstractpub_date
2011-04-19 00:00:00pages
3250-60issue
15eissn
0006-2960issn
1520-4995journal_volume
50pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全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...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi981838q
更新日期:1998-10-20 00:00:00
abstract::Kinetics studies in homogeneous aqueous solution showed that solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus (a bacterial DD-peptidase) was inhibited by the amphiphilic glycolipid antibiotic moenomycin. Inhibition at the peptidase site was determined by competition exp...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi982309p
更新日期:1999-08-10 00:00:00
abstract::The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi800134y
更新日期:2008-07-15 00:00:00
abstract::Many DNA polymerases select their natural substrates, deoxy- as opposed to ribonucleoside triphosphates, with a selectivity greater than 10000-fold. The function of a highly conserved residue, Tyr416, in the palm domain of the parental enzyme, an exo(-) derivative of RB69 DNA polymerase (gp43), a member of the pol alp...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0202171
更新日期:2002-08-13 00:00:00
abstract::Phosphodeoxyribomutase from Escherichia coli has been purified to homogeneity. Chromatography on DEAE-Sephadex revealed three peaks of enzyme activity, designated form I, form II, and form III. Form III could be further separated into form III-1 and form III-2 by polyacrylamide gel electrophoresis. The four different ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00674a021
更新日期:1975-02-11 00:00:00
abstract::Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy. The W57L mutant (retains tryptophan at residue 158) and the W158S m...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00228a008
更新日期:1991-04-09 00:00:00
abstract::Chemical and enzymatic probing (footprinting) of the reactivity of the promoter DNA backbone is applied to characterize two binary open complexes RPo1 (-Mg2+) and RPo2 (+Mg2+), formed by Escherichia coli RNA polymerase (E sigma 70) at the lambda PR promoter. We report that HO. detects major differences in backbone rea...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00048a004
更新日期:1995-12-05 00:00:00
abstract::The histone content of zinc-deficient (-Zn) Euglena gracilis decreases while, concomitantly, DNA content increases and the transcription rate is reduced markedly [Mazus, B., Falchuk, K. H., & Vallee, B. L. (1983) Biochemistry (in press); Falchuk, K. H., Fawcett, D. W., & Vallee, B. L. (1975) J. Cell Sci. 17, 57-78]. T...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00291a014
更新日期:1983-10-25 00:00:00
abstract::The fate of plasma and nuclear membrane polypeptides in preparations of acidic chromosomal protein from chicken erythrocytes has been investigated. It is shown that detergent extraction procedures (Nonidet P-40, Triton X-100, and saponin), commonly employed in the preparation of acidic chromosomal protein, cannot be r...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00670a034
更新日期:1976-12-14 00:00:00
abstract::Epoxides are cancer-causing agents chemically analogous to the nitrogen mustards, a family of powerful antitumor drugs. We found that the DNA interstrand cross-linking sequence preference of diepoxybutane is the same as that of the mustard mechlorethamine: 5'-GNC. Therefore, the genomic site of cross-linking alone can...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00059a034
更新日期:1993-03-02 00:00:00
abstract::Synthetic nonviral vector systems are attractive because of their apparent simplicity of preparation and use. However, there are many barriers to success at the moment, including the formulation of uniform and reproducible particles of transfection competent, condensed nucleic acids such as plasmid DNA (pDNA). For thi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi034325e
更新日期:2003-09-09 00:00:00
abstract::The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00234a032
更新日期:1991-05-21 00:00:00
abstract::Regions of the negative strands of the satellite RNAs of chicory yellow mottle virus (sCYMV1) and arabis mosaic virus (sArMV) have similarity in sequence and predicted secondary structure compared to the tobacco ringspot virus satellite RNA (sTRSV) hairpin ribozyme, suggesting that they may also be catalytic RNAs of a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00048a024
更新日期:1995-12-05 00:00:00
abstract::The molecular chaperone SecB binds to hydrophobic sections of unfolded secretory proteins and thereby prevents their premature folding prior to secretion by the translocase of Escherichia coli. Here, we have investigated the effect of the single-residue mutation of leucine 42 to arginine (L42R) centrally positioned in...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi902051e
更新日期:2010-03-23 00:00:00
abstract::Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi010283n
更新日期:2001-08-07 00:00:00
abstract::The phosphorylation mechanism of Dictyostelium discoideum nucleoside diphosphate (NDP) kinase was investigated by NMR. 31P chemical shifts were measured on both native and denatured enzyme. In the enzymatically phosphorylated enzyme denatured by 9 M urea or 7 M guanidine hydrochloride, the NDP kinase phosphohistidine ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00038a043
更新日期:1995-09-26 00:00:00
abstract::The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolut...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00284a021
更新日期:1983-07-19 00:00:00
abstract::The gene for the human substance P receptor (NK-1) was cloned using cDNA probes made by the polymerase chain reaction from primers based on the rat sequence. The gene spans 45-60 kb and is contained in five exons, with introns interrupting at sites homologous to those in the NK-2 receptor gene. Analysis of restriction...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00108a006
更新日期:1991-11-05 00:00:00
abstract::RNA:pseudouridine synthetase (Pus1) from Saccharomyces cerevisiae is a multisite specific enzyme that catalyzes the formation of pseudouridine at positions 34 and 36 of intron-containing precursor tRNAIle and at positions 27 and/or 28 of several yeast tRNAs. In this paper we demonstrate that the purified recombinant P...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972671o
更新日期:1998-05-19 00:00:00
abstract::We have employed a combination of gel retardation, protein-DNA cross-linking, and protein-protein cross-linking techniques to further examine the 2,3,7,8-tetrachlorodibenzo-p- dioxin-(TCDD-) dependent changes in the Ah receptor that result in a DNA-binding conformation. Gel retardation analysis of DNA-Sepharose chroma...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00225a026
更新日期:1991-03-19 00:00:00
abstract::A procedure for the preparation of coupling factor 1 (F1) from Escherichia coli lacking subunits delta and epsilon is described. Using chloroform and dimethyl sulfoxide, we can isolate F1 containing only subunits alpha, beta, and gamma [F1(alpha beta gamma)] directly from membrane vesicles in 10-mg quantities. Pure an...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00438a030
更新日期:1989-06-13 00:00:00
abstract::Surface-enhanced resonance Raman scattering (SERRS) spectra were obtained from carotenoids, in the all-trans configuration, located on the antenna complexes of Rhodobacter sphaeroides 2.4.1 membranes. Since resonance Raman (RR) spectra are barely detectable at the concentration that SERRS signals saturate, SERRS repre...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00455a017
更新日期:1990-01-23 00:00:00
abstract::Ultraviolet absorption techniques were used to study the thermodynamics of duplex formation for a DNA decamer, d(GCGAAAAGCG).d(CGCTTTTCGC), and a series of related duplexes, each of which contains a bulged base centered in the A.T tract. Thermodynamic parameters were obtained from nonlinear least-squares fits of the m...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00230a031
更新日期:1991-04-23 00:00:00
abstract::We have isolated from rabbit liver three cDNA clones of 1400-1800 base pairs that hybridize selectively to RNA from animals treated with phenobarbital. The nucleotide sequences of the cDNAs have been determined. In the protein coding region the nucleotide sequences of two of the cDNAs are 88% homologous, and the third...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00297a005
更新日期:1984-01-17 00:00:00
abstract::Separation of the gradient-purified gastric microsome into two membrane subfractions of distinct enzymatic and phospholipid composition has been achieved by mild SDS (0.033% w/v) treatment followed by sucrose gradient centrifugation of the pig and rabbit gastric microsomes. While the high-density membranes had all of ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00425a013
更新日期:1988-12-13 00:00:00
abstract::The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (Nqo1-Nqo14). Of these, seven subunits (Nqo7, Nqo8, and Nqo10-14) which are equivalent to the mitochondrial DNA-encoded subunits of complex I constitute the membrane segment of the enzyme compl...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi034166z
更新日期:2003-04-22 00:00:00
abstract::The orientation of eukaryotic cytochromes P450, with respect to the membrane of the endoplasmic reticulum, has been investigated. There is now good evidence that the tertiary structure of these proteins is essentially the same as that of the soluble bacterial isoenzyme cytochrome P450CI, with the exception of an exten...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00215a011
更新日期:1991-01-08 00:00:00
abstract::The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to re...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi001941r
更新日期:2001-02-20 00:00:00
abstract::Protein electrochemistry in an ultra-thin-layer electrochemical cell suitable for UV/vis and IR spectroscopy has been used to characterize the vibrational modes of the primary electron donors of Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers in their neutral and cation radical states (P and P+, ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00068a007
更新日期:1993-05-04 00:00:00
abstract::This work describes the biochemical characterization of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein (GAP) regulating the RAS gene products. A fragment of 383 residues (amino acids 1644-2026) was produced in Escherichia coli as glutathione S-transferase fusion protein (GST-Ira2p-...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00042a008
更新日期:1995-10-24 00:00:00