Abstract:
:Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acylation site, and catalytic activity can be inhibited by ligand binding to a peripheral site. The three-dimensional structures of AChE-ligand complexes reveal a narrow and deep active site gorge and indicate that ligands specific for the acylation site at the base of the gorge must first traverse the peripheral site near the gorge entrance. In recent studies attempting to clarify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slow the rates at which other ligands enter and exit the acylation site, a feature we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that cationic substrates can form a low-affinity complex at the peripheral site that accelerates catalytic hydrolysis at low substrate concentrations but results in substrate inhibition at high concentrations because of steric blockade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we demonstrate that a key residue in the human AChE peripheral site with which the substrate acetylthiocholine interacts is D74. We extend our kinetic model to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant K(S), from the dependence of the substrate hydrolysis rate on substrate concentration. For human AChE, a K(S) of 1.9+/-0.7 mM obtained by fitting this substrate inhibition curve agreed with a K(S) of 1.3+/-1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For Torpedo AChE, a K(S) of 0.5+/- 0.2 mM obtained from substrate inhibition agreed with a K(S) of 0.4+/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the K(S) to 4-10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While the turnover number k(cat) was unchanged in the human D74G mutant, the roughly 20-fold decrease in acetylthiocholine affinity for the peripheral site in D74G resulted in a corresponding decrease in k(cat)/K(app), the second-order hydrolysis rate constant, in the mutant. In addition, we show that D74 is important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This inhibitory effect, measured by the relative decrease in the first-order phosphorylation rate constant k(OP) for the neutral organophosphate 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC) that resulted from fasciculin binding, decreased from 0.002 in wild-type human AChE to 0.24 in the D74G mutant.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Mallender WD,Szegletes T,Rosenberry TLdoi
10.1021/bi000210osubject
Has Abstractpub_date
2000-07-04 00:00:00pages
7753-63issue
26eissn
0006-2960issn
1520-4995pii
bi000210ojournal_volume
39pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Using solution-state NMR spectroscopy, new insights into the early events governing amelogenin supramolecular self-assembly have been identified using sodium chloride and calcium chloride to trigger the association. Two-dimensional 1H-15N HSQC spectra were recorded for 15N- and 13C-labeled murine amelogenin as a funct...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi8018288
更新日期:2008-12-16 00:00:00
abstract::The binding of Lens culinaris lectin to the receptors of rabbit erythrocytes was studied by a quantitative fluorometric method using the fluoresceinated conjugate. Equilibrium data showed the presence of 1.2 x 10(6) receptors/cell with an association constant varying from 8 to 3 x 10(6) M-1 over a temperature range of...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00616a022
更新日期:1978-11-14 00:00:00
abstract::Biochemical experiments, using the well-defined human liver CYP2C9 expressed in yeast, and molecular modeling techniques were used to derive a predictive model for substrates of CYP2C9. The ability of 10 2-aroylthiophenes related to tienilic acid to act as substrates for CYP2C9 was studied. Four of them were original ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00033a007
更新日期:1995-08-22 00:00:00
abstract::Integrins are one of the major mediators of cellular adherence. Structurally the component alpha and beta chains are characterized by extensive intrachain disulfide bonding. The assignment of these bonds is currently based on homology with the chains of the integrin alphaIIbbeta3. However, recent crystallographic anal...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi034726u
更新日期:2003-11-11 00:00:00
abstract::The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein with the oxidizable Cys(56) replaced with Ser. The Nkx2.5 or Nkx2.5(C56S) homeodomain is 73% identical in sequence to and has the same NMR structure as the vnd (ventral nervous system defective)/NK-2 homeodomain of Drosophila when bound...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi050835s
更新日期:2005-09-20 00:00:00
abstract::Thiol dioxygenases are mononuclear non-heme FeII-dependent metalloenzymes that initiate the oxidative catabolism of thiol-containing substrates to their respective sulfinates. Cysteine dioxygenase (CDO), the best characterized mammalian thiol dioxygenase, contains a three-histidine (3-His) coordination environment rat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.0c00267
更新日期:2020-07-07 00:00:00
abstract::4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with [alpha-32P]azido-PPi in the dark under condition...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00452a021
更新日期:1989-12-26 00:00:00
abstract::A method is described for measuring equilibrium constants of DNA-protein interactions using gel chromatography. This technique has been used to study the sequence-specific interaction of the HinfI restriction endonuclease with DNA. HinfI has a monomeric molecular weight of 31000 and exists as a dimer in its active for...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00333a037
更新日期:1985-06-04 00:00:00
abstract::The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.6b01027
更新日期:2017-02-14 00:00:00
abstract::The growth and shortening of microtubules in guanosine triphosphate-(GTP-) mediated dynamic instability has previously been observed to occur at rates which are remarkably variable (Gildersleeve et al., 1992, Chrétien et al., 1995). Neighboring microtubules observed simultaneously can grow or shorten at different rate...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi961070e
更新日期:1996-11-12 00:00:00
abstract::The gamma-phosphoryl groups of two intermediates (M-ATP and M-ADP-P1) in the pathway of MgATP hydrolysis by myosin undergo extensive oxygen exchange with water. Actin activates the overall rate of hydrolysis at a rate-limiting step which follows these exchange reactions. Thus, actin, by decreasing the turnover time of...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00620a022
更新日期:1977-01-11 00:00:00
abstract::Mucopolysaccharides were isolated from calf cerebral microvasculature and calf aorta. The only complex carbohydrates that exhibited anticoagulant activity were heparin-like components. The biologic potencies of calf cerebral and aortic heparin-like species were 2.92 units/mg of anti-factor Xa activity and 2.85 units/m...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00303a023
更新日期:1984-04-10 00:00:00
abstract::The organization and interaction of chlorophylls (Chl) and the kinetics of the energy transfer in the core antenna of photosystem I (PSI) trimeric and monomeric complexes, isolated from Spirulina platensis with Triton X-100 have been studied by stationary and time-resolved fluorescence. At 295 K both complexes show an...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi970386z
更新日期:1997-11-11 00:00:00
abstract::Fluorescent derivatives of short oligonucleotides of defined sequence were prepared by the incorporation of 5-(propylamino)uridine via current phosphoramidite chemistry, followed by derivatization of the propylamine function with mansyl chloride. These oligomers, annealed to complementary oligomers, yielded short dupl...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00437a014
更新日期:1989-05-30 00:00:00
abstract::Two intermediates in the biosynthetic pathway to bacterial menaquinones are o-succinylbenzoic acid and 1,4-dihydroxy-2-naphthoic acid. Cell-free extracts have been prepared from Escherichia coli which catalyze the conversion of labeled o-succinylbenzoic acid to the naphthoic acid and also to menaquinones. The naphthoa...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00667a007
更新日期:1976-11-02 00:00:00
abstract::The kringle 2 (K2) module of human plasminogen (Pgn) binds L-lysine and analogous zwitterionic compounds, such as the antifibronolytic agent trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA). Far-UV CD and NMR spectra reveal little conformational change in K2 upon ligand binding. However, retarded (1)H-(2)H isotop...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9917378
更新日期:1999-11-30 00:00:00
abstract::Brief exposure of purple membrane (PM) to dilute Triton X-100 eliminates the actinic light effect on the relative amounts of fast M (Mf) and slow M (Ms) intermediates and alters the character and kinetics of the photocycle, without destroying the native BR trimers (Mukhopadhyay et al., 1994). Particular membrane lipid...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi960738m
更新日期:1996-07-16 00:00:00
abstract::For some hormone receptors, the early events of signal transduction depend on their molecular arrangement and interactions at the cell surface. An understanding of the mechanism of signal transduction in general needs a careful analysis of the receptor distribution. Here, we present the first quantitative measurement ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00124a004
更新日期:1992-03-10 00:00:00
abstract::Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer consisting of four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha, beta, and delta, the last being endogenous calmodulin). The enzyme is activated by a variety of effectors actin...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi982060b
更新日期:1999-02-23 00:00:00
abstract::Snapshots of three consecutive steps in the proteolytic reaction of HIV-1 protease (PR) were obtained in crystal structures at resolutions of 1.2-1.4 Å. Structures of wild-type protease and two mutants (PR(V32I) and PR(I47V)) with V32I and I47V substitutions, which are common in drug resistance, reveal the gem-diol te...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi3008092
更新日期:2012-10-02 00:00:00
abstract::The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (mi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9916675
更新日期:1999-11-30 00:00:00
abstract::Human cystathionine beta-synthase (CBS) is an essential enzyme for the removal of the toxic metabolite homocysteine. Heme and pyridoxal phosphate (PLP) cofactors are necessary to catalyze the condensation of homocysteine and serine to generate cystathionine. While the role for the PLP cofactor is thought to be similar...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0010874
更新日期:2001-01-16 00:00:00
abstract::Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structur...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi990286o
更新日期:1999-07-06 00:00:00
abstract::Glycophorin A is a sialoglycoprotein isolated from human erythrocyte membranes which seems to exist as stable dimeric complexes in the presence of sodium dodecyl sulfate. When analyzed by dodecyl sulfate acrylamide electrophoresis this molecule forms two PAS-stainable bands (PAS-U and PAS-2) which are reversibly inter...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00650a028
更新日期:1976-03-09 00:00:00
abstract::We report cloning, expression in Escherichia coli, and purification of cytochrome P450 from a deep-sea bacterium Photobacterium profundum strain SS9 (P450-SS9). The enzyme, which is predominately high spin (86%) in the absence of any added ligand, binds fatty acids and their derivatives and exhibits the highest affini...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi101466y
更新日期:2010-12-21 00:00:00
abstract::Transient contacts between denatured polypeptide chains are likely to play an important part in the initial stages of protein aggregation and fibrillation. To analyze the nature of such contacts, we have carried out a protein engineering study of the 102-residue protein U1A, which aggregates transiently in the wild-ty...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi048509k
更新日期:2004-10-19 00:00:00
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...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9702391
更新日期:1997-04-29 00:00:00
abstract::A series of human lymphoblastoid cell lines derived from nonleukemic donors are known to be cysteine prototrophs (cys+), while several lymphoblastoid lines derived from leukemic donors are cysteine auxotrophs (cys-). We have tested representative cell lines of each type for their content of cystathionase enzyme activi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00508a041
更新日期:1981-03-03 00:00:00
abstract::Photosynthetic water oxidation takes place at the Mn4CaO5 cluster in photosystem II. Around the Mn4CaO5 cluster, a hydrogen bond network is formed by several water molecules, including four water ligands. To clarify the role of this water network in the mechanism of water oxidation, we investigated the effects of the ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.5b01120
更新日期:2016-01-26 00:00:00
abstract::Wideline 2H NMR spectroscopy was used to assess the conformational and orientational effects of N-acetylneuraminic acid (NeuAc) (sialic acid) as a component of a particular oligosaccharide chain at a bilayer membrane surface. For this purpose, three glycosphingolipids, sharing a neutral core tetrasaccharide and differ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi952964m
更新日期:1996-04-16 00:00:00