Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis.

Abstract:

:Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-(delta-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K(d) values of 7.9, 6.9, and 0.28 microM, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K(d) values of 1.1 and 0.73 microM, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V(max)/K(m)) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Castro C,Gratson AA,Evans JC,Jiracek J,Collinsová M,Ludwig ML,Garrow TA

doi

10.1021/bi049821x

subject

Has Abstract

pub_date

2004-05-11 00:00:00

pages

5341-51

issue

18

eissn

0006-2960

issn

1520-4995

journal_volume

43

pub_type

杂志文章
  • Targeted cleavage of polynucleotides by complementary oligonucleotides covalently linked to iron-porphyrins.

    abstract::Oligothymidylates covalently linked to iron-porphyrins were synthesized to target the nuclease activity of Fe-porphyrin to complementary polynucleotides. In the presence of oxygen and a reducing agent, oligo(dT)7 bearing the reactive group attached to the 3'-phosphate was shown to be active in the cleavage of poly(dA)...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00370a002

    authors: Le Doan T,Perrouault L,Helene C,Chassignol M,Thuong NT

    更新日期:1986-11-04 00:00:00

  • Light-chain-independent binding of adaptors, AP180, and auxilin to clathrin.

    abstract::Binding of coated vesicle assembly proteins to clathrin causes it to assemble into regular coat structures. The assembly protein fraction of bovine brain coated vesicles comprises AP180, auxilin, and HA1 and HA2 adaptors. Clathrin heavy chains, separated from their light chains, polymerize with unimpaired efficiency w...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00101a027

    authors: Lindner R,Ungewickell E

    更新日期:1991-09-17 00:00:00

  • Characterization of recombinant Saccharomyces cerevisiae manganese-containing superoxide dismutase and its H30A and K170R mutants expressed in Escherichia coli.

    abstract::All known Mn-containing superoxide dismutases (MnSODs) have a highly conserved histidine (His-30 in Escherichia coli FeSOD) in the active-site channel, and nearly all have an active-site arginine (Arg-170) that has been proposed to play a combined structural and functional role [Chan et al., Arch. Biochem. Biophys. 27...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi980167t

    authors: Borders CL Jr,Bjerrum MJ,Schirmer MA,Oliver SG

    更新日期:1998-08-11 00:00:00

  • tRNA 3' processing in plants: nuclear and mitochondrial activities differ.

    abstract::The nuclear tRNA 3' processing activity from wheat has been characterized and partially purified. Several characteristics of the wheat nuclear 3' processing enzyme now allow this activity to be distinguished from its mitochondrial counterpart. The nuclear enzyme is an endonuclease, which we termed nuclear RNase Z. The...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi992253e

    authors: Mayer M,Schiffer S,Marchfelder A

    更新日期:2000-02-29 00:00:00

  • 13C NMR studies of methylene and methine carbons of substrate bound to a 280,000-dalton protein, porphobilinogen synthase.

    abstract::13C NMR has been used to observe the equilibrium complex of [5,5-2H,5-13C]-5-aminolevulinate [( 5,5-2H,5-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [5,5-2H,5-13C]ALA (chemical shift 46.9 ppm in D2O) was prepared from [5-13C]ALA through enolization in deu...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00412a039

    authors: Jaffe EK,Markham GD

    更新日期:1988-06-14 00:00:00

  • 1H NMR studies of tris(phenanthroline) metal complexes bound to oligonucleotides: structural characterizations via selective paramagnetic relaxation.

    abstract::The selective paramagnetic relaxation of oligonucleotide protons of d(GTGCAC)2 by delta- and lambda-Ni(phen)3(3+) and delta- and lambda-Cr(phen)3(3+) has been examined to obtain some structural insight into the noncovalent binding of tris(phenanthroline) metal complexes to DNA. The experiments demonstrate that the rel...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00459a007

    authors: Rehmann JP,Barton JK

    更新日期:1990-02-20 00:00:00

  • Kinetics of polymerization of a fluoresceinated derivative of complement protein C9 by the membrane-bound complex of complement proteins C5b-8.

    abstract::The fluorescence self-quenching by energy transfer of FITC-C9, a fluoresceinated derivative of human complement protein C9 [Sims, P.J. (1984) Biochemistry (preceding paper in this issue)], has been used to monitor the kinetics of C9 polymerization induced by the membrane-associated complex of complement proteins C5b-8...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00309a021

    authors: Sims PJ,Wiedmer T

    更新日期:1984-07-03 00:00:00

  • Ion concentration and temperature dependence of DNA binding: comparison of PurR and LacI repressor proteins.

    abstract::Purine repressor (PurR) binding to specific DNA is enhanced by complexing with purines, whereas lactose repressor (LacI) binding is diminished by interaction with inducer sugars despite 30% identity in their protein sequences and highly homologous tertiary structures. Nonetheless, in switching from low- to high-affini...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi0028643

    authors: Moraitis MI,Xu H,Matthews KS

    更新日期:2001-07-10 00:00:00

  • Extreme Expression of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Breast Cancer As Measured by Liquid Chromatography and Isotope Dilution Tandem Mass Spectrometry.

    abstract::Apurinic/apyrimidinic endonuclease 1 (APE1) is a DNA repair protein and plays other important roles. Increased levels of APE1 in cancer have been reported. However, available methods for measuring APE1 levels are indirect and not quantitative. We previously developed an approach using liquid chromatography and tandem ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.5b00928

    authors: Coskun E,Jaruga P,Reddy PT,Dizdaroglu M

    更新日期:2015-09-29 00:00:00

  • Transient kinetics of electron-transfer reactions of flavodoxins.

    abstract::Stopped-flow and laser photolysis methods have been used to investigate the rates of electron-transfer reactions of fully reduced riboflavin and the three oxidation states of Clostridium pasteurianum flavodoxin. Both normal and 7,8-dichloroflavin analogues were studied. Redox reagents included oxygen, ferricyanide, fe...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00521a005

    authors: Jung J,Tollin G

    更新日期:1981-09-01 00:00:00

  • Copper binding to beta-2-microglobulin and its pre-amyloid oligomers.

    abstract::Beta-2-microglobulin (beta2m) deposits as amyloid fibrils in the musculoskeletal system of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that Cu(II) binding causes beta2m to organize into nativelike dimers and tetramers that precede amyloid formation. Cu(II) is...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi901172y

    authors: Srikanth R,Mendoza VL,Bridgewater JD,Zhang G,Vachet RW

    更新日期:2009-10-20 00:00:00

  • Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation.

    abstract::Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00342a041

    authors: Kedersha NL,Tkacz JS,Berg RA

    更新日期:1985-10-08 00:00:00

  • Probing the "charge cluster mechanism" in amphipathic helical cationic antimicrobial peptides.

    abstract::Clustering of anionic lipids away from zwitterionic ones by cationic antimicrobial agents has recently been established as a mechanism of action of natural small, flexible peptides as well as non-natural synthetic peptide mimics. One of the largest classes of antimicrobial peptides consists of peptides that form catio...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi100378m

    authors: Epand RF,Maloy WL,Ramamoorthy A,Epand RM

    更新日期:2010-05-18 00:00:00

  • A mechanistic investigation of the C-terminal redox motif of thioredoxin reductase from Plasmodium falciparum.

    abstract::High-molecular mass thioredoxin reductases (TRs) are pyridine nucleotide disulfide oxidoreductases that catalyze the reduction of the disulfide bond of thioredoxin (Trx). Trx is responsible for reducing multiple protein disulfide targets in the cell. TRs utilize reduced β-nicotinamide adenine dinucleotide phosphate to...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi400931k

    authors: Snider GW,Dustin CM,Ruggles EL,Hondal RJ

    更新日期:2014-01-28 00:00:00

  • Physical behavior of the hydrophobic core of membranes: properties of 1-stearoyl-2-linoleoyl-sn-glycerol.

    abstract::Phospholipids containing a saturated fatty acid in the primary position and an unsaturated fatty acid in the secondary position are a major structural part of biological membranes. The mixed-chain hydrophobic core of the membranes is the diacylglycerol part. To better understand the core properties of membranes we hav...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00051a015

    authors: Di L,Small DM

    更新日期:1995-12-26 00:00:00

  • Sequence context- and temperature-dependent nucleotide excision repair of a benzo[a]pyrene diol epoxide-guanine DNA adduct catalyzed by thermophilic UvrABC proteins.

    abstract::The influence of DNA base sequence context on the removal of a bulky benzo[a]pyrene diol epoxide-guanine adduct, (+)-trans-B[a]P-N2-dG (G*), by UvrABC nuclease from the thermophilic organism Bacillus caldotenax was investigated. The lesion was flanked by either T or C in otherwise identical complementary 43-mer duplex...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi700294k

    authors: Ruan Q,Liu T,Kolbanovskiy A,Liu Y,Ren J,Skorvaga M,Zou Y,Lader J,Malkani B,Amin S,Van Houten B,Geacintov NE

    更新日期:2007-06-12 00:00:00

  • Role of the structural domain of troponin C in muscle regulation: NMR studies of Ca2+ binding and subsequent interactions with regions 1-40 and 96-115 of troponin I.

    abstract::The interaction between the calcium binding and inhibitory components of troponin is central to the regulation of muscle contraction. In this work, two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (2D-¿1H,15N¿-HSQC NMR) spectroscopy was used to determine the stoichiometry, affinity, an...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi992579n

    authors: Mercier P,Li MX,Sykes BD

    更新日期:2000-03-21 00:00:00

  • Mapping the suramin-binding sites of human neutrophil elastase: investigation by fluorescence resonance energy transfer and molecular modeling.

    abstract::Neutrophil elastase (NE), a mediator of inflammation, binds with high affinity numerous anionic molecules including suramin, a polysulfated naphthylurea, which inhibits it with a Ki of 0.2 microM and a 4:1 suramin:NE stoichiometry and thus constitutes a potential therapeutic agent. In an attempt to locate the suramin ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi971029r

    authors: Mély Y,Cadène M,Sylte I,Bieth JG

    更新日期:1997-12-16 00:00:00

  • Proximity of periplasmic loops in the lactose permease of Escherichia coli determined by site-directed cross-linking.

    abstract::Out of over 60 single-Cys mutants in putative periplasmic loops in lactose permease, three mutants [Tyr101 --> Cys (loop III/IV), Leu313 --> Cys (loop IX/X), and Ser375 --> Cys (loop XI/XII)] spontaneously form disulfide-linked dimers, indicating that these loops are located on the periphery of the 12-helix bundle tha...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi971172k

    authors: Sun J,Kaback HR

    更新日期:1997-09-30 00:00:00

  • Mobilities of the inner three core residues and the Man(alpha 1--6) branch of the glycan at Asn78 of the alpha-subunit of human chorionic gonadotropin are restricted by the protein.

    abstract::Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone involved in the maintenance of the corpus luteum in early pregnancy. The free alpha-subunit of hCG has a biological activity of its own, namely, stimulation of prolactin secretion from term pregnancy decidual cells [Blithe, D. L., et al. (1991)...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi9718548

    authors: van Zuylen CW,de Beer T,Leeflang BR,Boelens R,Kaptein R,Kamerling JP,Vliegenthart JF

    更新日期:1998-02-17 00:00:00

  • Functional segregation of a predicted "hinge" site within the beta-strand linkers of Escherichia coli leucyl-tRNA synthetase.

    abstract::Some aminoacyl-tRNA synthetases (AARSs) employ an editing mechanism to ensure the fidelity of protein synthesis. Leucyl-tRNA synthetase (LeuRS), isoleucyl-tRNA synthetase (IleRS), and valyl-tRNA synthetase (ValRS) share a common insertion, called the CP1 domain, which is responsible for clearing misformed products. Th...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi702494q

    authors: Mascarenhas AP,Martinis SA

    更新日期:2008-04-22 00:00:00

  • NG-methyl-L-arginine functions as an alternate substrate and mechanism-based inhibitor of nitric oxide synthase.

    abstract::NG-Methyl-L-arginine (L-NMA) is one of the most commonly used inhibitors of the nitric oxide synthases (NOS). Results reported here demonstrate that L-NMA is an alternate substrate and a mechanism-based inhibitor of the inducible NOS purified from murine macrophages. The irreversible inhibition displays pseudo-first-o...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00088a020

    authors: Olken NM,Marletta MA

    更新日期:1993-09-21 00:00:00

  • NMR structure of navel orangeworm moth pheromone-binding protein (AtraPBP1): implications for pH-sensitive pheromone detection.

    abstract::The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through the aqueous s...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi9020132

    authors: Xu X,Xu W,Rayo J,Ishida Y,Leal WS,Ames JB

    更新日期:2010-02-23 00:00:00

  • Protein arginine deiminase 4: evidence for a reverse protonation mechanism.

    abstract::The presumed role of an overactive protein arginine deiminase 4 (PAD4) in the pathophysiology of rheumatoid arthritis (RA) suggests that PAD4 inhibitors could be used to treat an underlying cause of RA, potentially offering a mechanism to stop further disease progression. Thus, the development of such inhibitors is of...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi700095s

    authors: Knuckley B,Bhatia M,Thompson PR

    更新日期:2007-06-05 00:00:00

  • Interactions of the antimicrobial peptides temporins with model biomembranes. Comparison of temporins B and L.

    abstract::Temporins are short (10-13 amino acids) and linear antimicrobial peptides first isolated from the skin of the European red frog, Rana temporaria, and are effective against Gram-positive bacteria and Candida albicans. To get insight into their mechanism(s) of action, we compared the effects on model membranes exerted b...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi011929e

    authors: Zhao H,Rinaldi AC,Di Giulio A,Simmaco M,Kinnunen PK

    更新日期:2002-04-02 00:00:00

  • Specific RNA binding by Q beta coat protein.

    abstract::The interaction between the bacteriophage Q beta coat protein and its specific binding site on Q beta genomic RNA was characterized by using a nitrocellulose filter binding assay. Q beta coat protein bound to a synthetic 29-nucleotide RNA hairpin with an association constant of 400 microM-1 at 4 degrees C, 0.2 M ionic...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00427a011

    authors: Witherell GW,Uhlenbeck OC

    更新日期:1989-01-10 00:00:00

  • Intramolecular disulfide loop formation in a peptide containing two cysteines.

    abstract::The cyanogen bromide fragment comprising residues 115-181 of Kunitz soybean trypsin inhibitor is a soluble random-coil peptide at pH 7 containing two cysteines separated by eight other amino acids in the primary sequence. Four of the six rate constants have been determined for the three disulfide exchange reactions be...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00377a005

    authors: Snyder GH

    更新日期:1987-02-10 00:00:00

  • Probing the nucleotide binding sites of axonemal dynein with the fluorescent nucleotide analogue 2'(3')-O-(-N-Methylanthraniloyl)-adenosine 5'-triphosphate.

    abstract::MantATP [2'(3')-O-(-N-methylanthraniloyl)-adenosine 5'-triphosphate] was employed as a fluorescence probe of the nucleotide-binding sites of dynein from sea urchin sperm flagella. MantATP binds specifically with enhanced fluorescence (approximately 2.2-fold), homogeneous lifetime (8.4 ns), and high anisotropy (r appro...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi9730184

    authors: Mocz G,Helms MK,Jameson DM,Gibbons IR

    更新日期:1998-07-07 00:00:00

  • Distal pocket polarity in ligand binding to myoglobin: structural and functional characterization of a threonine68(E11) mutant.

    abstract::Site-directed mutagenesis studies have confirmed that the distal histidine in myoglobin stabilizes bound O2 by hydrogen bonding and have suggested that it is the polar character of the imidazole side chain rather than its size that limits the rate of ligand entry into the protein. We constructed an isosteric Val68 to ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00239a025

    authors: Smerdon SJ,Dodson GG,Wilkinson AJ,Gibson QH,Blackmore RS

    更新日期:1991-06-25 00:00:00

  • CO photodissociation dynamics in cytochrome P450BM3 studied by subpicosecond visible and mid-infrared spectroscopy.

    abstract::Cytochrome P450BM3 is a bacterial enzyme with a heme cofactor that binds small diatomic ligands. Here we report the first study of carbon monoxide (CO) photodissociation and rebinding in ferrous P450BM3 on an ultrafast time scale. We monitored dissociation of carbon monoxide upon Soret band excitation using visible an...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi900351m

    authors: Rupenyan A,Commandeur J,Groot ML

    更新日期:2009-07-07 00:00:00