Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules. Pre-steady-state kinetic experiments have been employed to investigate the cooperative interactions between the motor domains of the MC1 dimer and to establish the ATPase mechanism. Our results indicate that the active sites of dimeric Ncd free in ... More
Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd.
AuthorsMackey AT, Gilbert SP
JournalBiochemistry
PubMed ID10684615
'Ncd is a minus-end-directed microtubule motor and a member of the kinesin superfamily. The Ncd dimer contains two motor domains, and cooperative interactions between the heads influence the interactions of each respective motor domain with the microtubule. The approach we have taken to understand the cooperativity between the two motor ... More
Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein.
AuthorsGalletto R, Rajendran S, Bujalowski W
JournalBiochemistry
PubMed ID11041861
'Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and ... More
The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity.
AuthorsMurphy CT, Spudich JA
JournalBiochemistry
PubMed ID10090768
'We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ... More
Kinetic mechanism of kinesin motor domain.
AuthorsMa YZ, Taylor EW
JournalBiochemistry
PubMed ID7548087
'The kinetic mechanism of the human kinesin ATPase motor domain K379, expressed in Escherichia coli, was determined by transient and steady-state kinetic studies. The steps in nucleotide binding were measured using the fluorescent substrate analogues, methylanthraniloyl ATP (mant-ATP) and mant-ADP. Both nucleotides gave a two-step fluorescence signal, an increase followed ... More
An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex.
AuthorsZhuang Z, Berdis AJ, Benkovic SJ
JournalBiochemistry
PubMed ID16800623
'In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form ... More
Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase.
AuthorsOstap EM, Pollard TD
JournalJ Cell Biol
PubMed ID8601584
'Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench ... More
'While most of the sequence of myosin''s motor domain is highly conserved among various organisms and tissue types, the junctions between the 25 and 50 kDa domains and the 50 and 20 kDa domains are strikingly divergent. The 50-20K loop is positioned to interact with actin, while the 25-50K loop ... More
Kinetic studies of dimeric Ncd: evidence that Ncd is not processive.
AuthorsFoster KA, Gilbert SP
JournalBiochemistry
PubMed ID10677228
'Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. ... More
Binding of steroid modulators to recombinant cytosolic domain from mouse P-glycoprotein in close proximity to the ATP site.
AuthorsDayan G, Jault JM, Baubichon-Cortay H, Baggetto LG, Renoir JM, Baulieu EE, Gros P, Di Pietro A
JournalBiochemistry
PubMed ID9398248
'We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395-581 of mouse mdr1 P-glycoprotein bound fluorescent 2''(3'')-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity [Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J. -C., Deléage, G., & Di Pietro, A. (1996) J. Biol. Chem. 271, 11652-11658]. The present work shows that a ... More
Inhibition of ATPase activity of the recA protein by ATP ribose-modified analogs.
AuthorsKarasaki Y, Higashi K
JournalArch Biochem Biophys
PubMed ID6237610
'The single-stranded, DNA-dependent ATPase activity of purified recA protein was found to be inhibited competitively by ribose-modified analogs of ATP, 3''-O-anthraniloyl-ATP (Ant-ATP), and 3''-O-(N-methylanthraniloyl)-ATP (Mant-ATP). The Ki values for Ant-ATP and Mant-ATP were around 7 and 3 microM at pH 7.5, respectively. The inhibitions by these analogs were much stronger ... More
Loop I can modulate ADP affinity, ATPase activity, and motility of different scallop myosins. Transient kinetic analysis of S1 isoforms.
AuthorsKurzawa-Goertz SE, Perreault-Micale CL, Trybus KM, Szent-Györgyi AG, Geeves MA
JournalBiochemistry
PubMed ID9585566
'The striated muscle myosin of Placopecten moves actin faster in in vitro motility assays and has a higher actin-activated ATPase turnover rate than the myosin of the catch muscle. The heavy chain sequences of the two PlacoS1s are almost identical except at the surface loop 1 near the nucleotide binding ... More
Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein.
AuthorsBooth CL, Pulaski L, Gottesman MM, Pastan I
JournalBiochemistry
PubMed ID10820025
'Human P-glycoprotein, the MDR1 gene product, requires both Mg(2+)-ATP binding and hydrolysis to function as a drug transporter; however, the mechanism(s) defining these events is not understood. In the present study, we explored the nature of Mg(2+)-ATP binding in the N-terminal nucleotide-binding domain of human P-glycoprotein and identified the minimal ... More
Unactivated PKR exists in an open conformation capable of binding nucleotides.
AuthorsLemaire PA, Tessmer I, Craig R, Erie DA, Cole JL
JournalBiochemistry
PubMed ID16866353
'The dsRNA-activated protein kinase, PKR, plays a pivotal role in the cellular antiviral response. PKR contains an N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain. An autoinhibition model has been proposed in which latent PKR exists in a closed conformation where the substrate binding cleft of the kinase ... More
Structural and kinetic studies of phosphorylation-dependent regulation in smooth muscle myosin.
AuthorsRosenfeld SS, Xing J, Cheung HC, Brown F, Kar S, Sweeney HL
JournalJ Biol Chem
PubMed ID9786863
'In this study, we have examined the mechanism of phosphorylation-dependent regulation in smooth muscle myosin through the use of structural and kinetic methodologies applied to several myosin fragments. Fluorescence anisotropy decay measurements demonstrate that regulatory light chain phosphorylation significantly reduces the rotational correlation time of regulatable myosin preparations, whereas minimally ... More
X-ray crystal structure and solution fluorescence characterization of Mg.2'(3')-O-(N-methylanthraniloyl) nucleotides bound to the Dictyostelium discoideum myosin motor domain.
AuthorsBauer CB, Kuhlman PA, Bagshaw CR, Rayment I
JournalJ Mol Biol
PubMed ID9405148
'Mant (2''(3'')-O-(N-methylanthraniloyl)) labeled nucleotides have proven to be useful tools in the study of the kinetic mechanism of the myosin ATPase by fluorescence spectroscopy. The sensitivity of the mant fluorophore to its local environment also makes it suitable to investigate the exposure of bound nucleotides to solvent from collisional quenching ... More
Nucleotide-dependent conformational changes in the sigma54-dependent activator DctD.
AuthorsWang YK, Park S, Nixon BT, Hoover TR
JournalJ Bacteriol
PubMed ID14526036
'Activators of sigma(54)-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open promoter complex. DctD(Delta1-142), a truncated and constitutively active form of the sigma(54)-dependent activator DctD from Sinorhizobium meliloti, displayed an altered DNase I footprint at its binding site located upstream of the dctA promoter in the presence of ... More
Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain.
AuthorsShimizu T, Sablin E, Vale RD, Fletterick R, Pechatnikova E, Taylor EW
JournalBiochemistry
PubMed ID7548090
'ncd is a kinesin-related motor protein from Drosophila that moves in the opposite direction along microtubules to kinesin. To learn more about the ncd mechanism, ncd motor domain (R335-K700) was expressed in Escherichia coli and its enzymatic characteristics were studied. The ncd motor domain was purified from the cell lysate ... More
A biosensor for fluorescent determination of ADP with high time resolution.
AuthorsKunzelmann S, Webb MR,
JournalJ Biol Chem
PubMed ID19801632
'Nearly every cellular process requires the presence of ATP. This is reflected in the vast number of enzymes like kinases or ATP hydrolases, both of which cleave the terminal phosphate from ATP, thereby releasing ADP. Despite the fact that ATP hydrolysis is one of the most fundamental reactions in biological ... More
'Hsp90 is one of the most abundant proteins in the cytosol of eukaryotic cells. Under physiological conditions Hsp90 has been shown to play a major role in several specific signaling pathways, including maturation of various kinases and maintenance of steroid receptors in an activable state. It is well established that ... More
Interacting head mechanism of microtubule-kinesin ATPase.
AuthorsMa YZ, Taylor EW
JournalJ Biol Chem
PubMed ID8995356
'Kinetic and equilibrium properties are compared for a monomeric kinesin construct (K332) and a dimeric construct (K379). MtK379 has a low affinity (5 x 10(4) M(-1)) and a high affinity (5 x 10(6) M(-1)) binding site for mant ADP while MtK332 has a single low affinity site (5 x 10(4) ... More
The conformation of the active site of myosin probed using mant-nucleotides.
AuthorsFranks-Skiba K, Cooke R
JournalBiophys J
PubMed ID7787057
'Changes in the conformation of the active site of myosin subfragment-1 (S1) may be linked to the production of force during the powerstroke. We probed the conformation of the nucleotide pocket by measuring the solvent accessibility of bound mant-nucleotides. Solvent accessibility was determined by measuring the quenching of fluorescence produced ... More
Kinetic characterization of a monomeric unconventional myosin V construct.
AuthorsTrybus KM, Krementsova E, Freyzon Y
JournalJ Biol Chem
PubMed ID10488077
'An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 ... More
Prenyl-flavonoids as potent inhibitors of the Pdr5p multidrug ABC transporter from Saccharomyces cerevisiae.
AuthorsConseil G, Decottignies A, Jault JM, Comte G, Barron D, Goffeau A, Di Pietro A
JournalBiochemistry
PubMed ID10841772
'The Pdr5p multidrug ABC ("ATP-binding cassette) transporter was highly overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3 gain-of-function mutation in the transcription factor-encoding gene PDR1 and disruption of genes encoding other plasma membrane ABC transporters. Solubilized and purified Pdr5p displayed a tryptophan-characteristic intrinsic fluorescence, whose quenching was ... More
Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer.
AuthorsSun M, Oakes JL, Ananthanarayanan SK, Hawley KH, Tsien RY, Adams SR, Yengo CM
JournalJ Biol Chem
PubMed ID16377637
'The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding ... More
New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes.
AuthorsHiratsuka T
JournalBiochim Biophys Acta
PubMed ID6132622
'The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3''-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence ... More
A kinetic study of the kinesin ATPase.
AuthorsSadhu A, Taylor EW
JournalJ Biol Chem
PubMed ID1534560
'The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2''-(3'')-O-(N-methylanthraniloyl)-ATP and mant-ADP. ... More
Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk.
AuthorsShaffer J, Sun G, Adams JA
JournalBiochemistry
PubMed ID11551213
'The COOH-terminal Src kinase (Csk) regulates a broad array of cellular processes via the specific phosphorylation and downregulation of Src family protein kinases. While Csk has been a topic for steady-state kinetic studies, the individual steps associated with substrate phosphorylation have not been investigated. To understand active-site phenomena, pre-steady-state and ... More
'Steady state measurements of the ATP turnover rate of myosin crossbridges in relaxed living mammalian muscle or in in vitro systems are complicated by other more rapid ATPase activities. To surmount these problems we have developed a technique to measure the nucleotide turnover rate of fully relaxed myosin heads in ... More
Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies.
AuthorsJekabsons MB, Echtay KS, Brand MD
JournalBiochem J
PubMed ID12030845
'Experiments were performed to test the hypothesis that recombinant human uncoupling protein-2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotide-inhibited uncoupling that is observed in kidney mitochondria. For this, sarkosyl-solubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and ... More
A structural change in the kinesin motor protein that drives motility.
AuthorsRice S, Lin AW, Safer D, Hart CL, Naber N, Carragher BO, Cain SM, Pechatnikova E, Wilson-Kubalek EM, Whittaker M, Pate E, Cooke R, Taylor EW, Milligan RA, Vale RD
JournalNature
PubMed ID10617199
'Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region ... More
Millisecond time resolution electron cryo-microscopy of the M-ATP transient kinetic state of the acto-myosin ATPase.
AuthorsWalker M, Trinick J, White H
JournalBiophys J
PubMed ID7787114
'The structure of the AM-ATP transient kinetic state of the acto-myosin ATPase cycle has been examined by electron microscopy using frozen-hydrated specimens prepared in low ionic strength. By spraying grids layered with the acto-S1 complex with ATP immediately before freezing, it was possible to examine the structure of the ternary ... More
Fluorescence changes of a label attached near the myosin active site on nucleotide binding in rat skeletal muscle fibres.
AuthorsFujita S, Nawata T, Yamada K
JournalJ Physiol
PubMed ID10066911
'1. Trinitrophenyl AMP (TNP-AMP) in the concentration range 10-300 microM induced an increase in fluorescence intensity at around 530 nm in skinned skeletal muscle fibres freshly obtained from rat psoas muscle. 2. The fluorescence intensity of the fibres depended on TNP-AMP concentration up to approximately 200 microM. The Kd of ... More
Interaction of mant-adenosine nucleotides and magnesium with kinesin.
AuthorsCheng JQ, Jiang W, Hackney DD
JournalBiochemistry
PubMed ID9548760
'Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluorescent transient. The pH and microtubule dependence of the rates and amplitudes indicates that the two phases are produced by release of bound mant-ADP, with an excess of the 3''-isomer, followed by ... More
Misactivated amino acids translocate at similar rates across surface of a tRNA synthetase.
AuthorsNomanbhoy TK, Schimmel PR
JournalProc Natl Acad Sci U S A
PubMed ID10792042
'Certain aminoacyl-tRNA synthetases have a second active site that destroys (by hydrolysis) errors of amino acid activation. For example, isoleucyl-tRNA synthetase misactivates valine (to produce valyl adenylate or Val-tRNA(Ile)) at its active site. The misactivated amino acid is then translocated to an editing site located >25 A away. The role ... More
Fluorescent nucleotide analogs: synthesis and applications.
AuthorsJameson DM, Eccleston JF
JournalMethods Enzymol
PubMed ID9170323
Synthesis of novel fluorescent-labelled dinucleoside polyphosphates.
AuthorsWright M, Miller AD
JournalBioorg Med Chem Lett
PubMed ID15125938
A novel tandem synthetic-biosynthetic procedure is described for the synthesis of four new fluorescent dinucleoside polyphosphates: mant-Ap4A, mant-AppCH2ppA, TNP-Ap4A and TNP-AppCH2ppA. These compounds are expected to supplement the existing etheno (epsilon) and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) labelled derivatives, being the fluorescent probes of choice to investigate polyphosphate/enzyme binding behaviour. ... More
ADP binding induces an asymmetry between the heads of unphosphorylated myosin.
AuthorsBerger CE, Fagnant PM, Heizmann S, Trybus KM, Geeves MA
JournalJ Biol Chem
PubMed ID11301326
Light chain phosphorylation is the key event that regulates smooth and non-muscle myosin II ATPase activity. Here we show that both heads of smooth muscle heavy meromyosin (HMM) bind tightly to actin in the absence of nucleotide, irrespective of the state of light chain phosphorylation. In striking contrast, only one ... More
Sea urchin axonemal motion supported by fluorescent, ribose-modified analogues of ATP.
AuthorsOmoto CK
JournalJ Muscle Res Cell Motil
PubMed ID1491072
The axonemal motion supported by fluorescent ribose-modified analogues, anthraniloyl ATP (Ant-ATP) and methylanthraniloyl ATP (Mant-ATP), was investigated. Ant-ATP and Mant-ATP supported good vigorous motion. A detailed study of the movement shows that the maximum beat frequencies (Vmax) were significantly lower with the analogues. However, Michaelis constants (Km) for beat frequency ... More
Interactions of cyclins with cyclin-dependent kinases: a common interactive mechanism.
AuthorsHeitz F, Morris MC, Fesquet D, Cavadore JC, Dorée M, Divita G
JournalBiochemistry
PubMed ID9125522
The formation of cdk-cyclin complexes has been investigated at the molecular level and quantified using spectroscopic approaches. In the absence of phosphorylation, cdk2, cdc2, and cdk7 form highly stable complexes with their "natural" cyclin partners with dissociation constants in the nanomolar range. In contrast, nonphosphorylated cdc2-cyclin H, cdk2-cyclin H, and ... More
The ATPase cross-bridge cycle of the Kar3 motor domain. Implications for single head motility.
AuthorsMackey AT, Gilbert SP
JournalJ Biol Chem
PubMed ID12446697
Kar3 is a minus-end directed microtubule motor involved in meiosis and mitosis in Saccharomyces cerevisae. Unlike Drosophila Ncd, the other well characterized minus-end directed motor that is a homodimer, Kar3 is a heterodimer with a single motor domain and either the associated polypeptides Cik1 or Vik1. Our mechanistic studies with ... More
A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor.
AuthorsBrendza KM, Sontag CA, Saxton WM, Gilbert SP
JournalJ Biol Chem
PubMed ID10767290
Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr(291) ... More
Kinetic mechanism and regulation of myosin VI.
AuthorsDe La Cruz EM, Ostap EM, Sweeney HL
JournalJ Biol Chem
PubMed ID11423557
Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine ... More
The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis.
AuthorsVertommen D, Bertrand L, Sontag B, Di Pietro A, Louckx MP, Vidal H, Hue L, Rider MH
JournalJ Biol Chem
PubMed ID8663445
All known 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX4GK(S/T)) in the 6-phosphofructo-2-kinase domain corresponding to the so-called nucleotide binding fold signature or Walker A motif. Mutagenesis and crystal structure data from several nucleotide binding proteins, which also contain this sequence, showed the importance of the lysine and serine/threonine residues in nucleotide ... More
Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy.
AuthorsSorensen CE, Novak I
JournalJ Biol Chem
PubMed ID11387334
The energy providing substrate ATP can be released from various cells and act extracellularly to regulate the same cells or neighboring cells. However, the pathway for ATP release and the eliciting physiological stimulus are unclear. Recently, we showed that ATP activates P2X and P2Y purinergic receptors on pancreatic ducts. Thus, ... More
Kinesin has three nucleotide-dependent conformations. Implications for strain-dependent release.
AuthorsXing J, Wriggers W, Jefferson GM, Stein R, Cheung HC, Rosenfeld SS
JournalJ Biol Chem
PubMed ID10852922
Although crystallographic information is available on several nucleotide-induced states in myosin, little is known about the corresponding structural changes in kinesin, since a crystallographic model is only available for the kinesin:ADP complex. This makes it difficult to characterize at a molecular level the structural changes that occur in this motor ... More
Correlation between the affinity of flavonoids binding to the cytosolic site of Leishmania tropica multidrug transporter and their efficiency to revert parasite resistance to daunomycin.
AuthorsPérez-Victoria JM, Chiquero MJ, Conseil G, Dayan G, Di Pietro A, Barron D, Castanys S, Gamarro F
JournalBiochemistry
PubMed ID10026252
The C-terminal nucleotide-binding domain (NBD2) of a P-glycoprotein-like transporter, encoded by the ltrmdr1 gene in Leishmania tropica and involved in parasite multidrug resistance (MDR), was overexpressed in Escherichia coli as a hexahistidine tagged protein and purified. The L. tropica recombinant domain efficiently bound fluorescent derivatives of ATP, the hydrophobic steroid ... More
The Escherichia coli Rho protein uses the energy of ATP binding and hydrolysis to translocate along RNA and cause transcription termination. Using fluorescence stopped-flow kinetic studies, we have discerned the conformational changes in the Rho protein that occur upon nucleotide and nucleic acid binding. We show that the 2', (3')-O-[N-methylanthraniloyl] ... More
Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity.
AuthorsBrown NR, Noble ME, Lawrie AM, Morris MC, Tunnah P, Divita G, Johnson LN, Endicott JA
JournalJ Biol Chem
PubMed ID10085115
We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 ... More
Interaction of actin and ADP with the head domain of smooth muscle myosin: implications for strain-dependent ADP release in smooth muscle.
AuthorsCremo CR, Geeves MA
JournalBiochemistry
PubMed ID9485324
Transient kinetic methods were used to study interactions between actin, MgADP, and smooth muscle (chicken gizzard) myosin subfragment 1 (smS1). The equilibrium dissociation constant (Kd) of actin for smS1 was 3.5 nM, tighter than that of skeletal S1 (skS1). Actin binding to smS1 was weakened 5-fold by saturation with ADP ... More
Tryptophan 512 is sensitive to conformational changes in the rigid relay loop of smooth muscle myosin during the MgATPase cycle.
AuthorsYengo CM, Chrin LR, Rovner AS, Berger CL
JournalJ Biol Chem
PubMed ID10827189
To examine the structural basis of the intrinsic fluorescence changes that occur during the MgATPase cycle of myosin, we generated three mutants of smooth muscle myosin motor domain essential light chain (MDE) containing a single conserved tryptophan residue located at Trp-441 (W441-MDE), Trp-512 (W512-MDE), or Trp-597 (W597-MDE). Although W441- and ... More
Kinetic mechanism of a monomeric kinesin construct.
AuthorsMa YZ, Taylor EW
JournalJ Biol Chem
PubMed ID8995355
The kinetic mechanism is analyzed for a monomeric human kinesin construct K332. In the absence of microtubules, the rate constants of the ATPase cycle are very similar to dimeric human kinesin K379 and whole kinesin from bovine brain. The microtubule-activated ATPase is 60 s(-1) at 20 degrees C; Km(Mt) is ... More
Kinetic and spectroscopic characterization of fluorescent ribose-modified ATP analogs upon interaction with skeletal muscle myosin subfragment 1.
The interaction of fluorescent ATP analog 2'(3')-O-[N-[2-[3-(5-fluoresceinyl)thioureido]-ethyl]carbamoyl]adenosine 5'-triphosphate (FEDA-ATP) with rabbit skeletal myosin subfragment 1 (S1) and acto-S1 was studied. This and related ATP analogs are potentially useful for determination of the ATPase activity of single myosin filaments using fluorescence microscopy [Sowerby et al. (1993) J. Mol. Biol. 234, 114-123]. ... More
Molecular properties of purified human uncoupling protein 2 refolded from bacterial inclusion bodies.
AuthorsJekabsons MB, Echtay KS, Arechaga I, Brand MD
JournalJ Bioenerg Biomembr
PubMed ID14740889
One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments ... More
Kinetic characterization of myosin head fragments with long-lived myosin.ATP states.
AuthorsFriedman AL, Geeves MA, Manstein DJ, Spudich JA
JournalBiochemistry
PubMed ID9657680
We have separately expressed the Dictyosteliumdiscoideum myosin II nonhydrolyzer point mutations E459V and E476K [Ruppel, K. M., and Spudich, J. A. (1996) Mol. Biol. Cell 7, 1123-1136] in the soluble myosin head fragment M761-1R [Anson et al. (1996) EMBO J. 15, 6069-6074] and performed transient kinetic analyses to characterize the ... More
Stabilization of the actomyosin complex by negative charges on myosin.
AuthorsFurch M, Remmel B, Geeves MA, Manstein DJ
JournalBiochemistry
PubMed ID10995227
Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideummyosin II residues D530, E531, and ... More
Transient kinetic analysis of the 130-kDa myosin I (MYR-1 gene product) from rat liver. A myosin I designed for maintenance of tension?
AuthorsColuccio LM, Geeves MA
JournalJ Biol Chem
PubMed ID10419463
The 130-kDa myosin I (MI(130)), product of the myr-1 gene, is one member of the mammalian class I myosins, a group of small, calmodulin-binding mechanochemical molecules of the myosin superfamily that translocate actin filaments. Roles for MI(130) are unknown. Our hypothesis is that, as with all myosins, MI(130) is designed ... More
Tight binding of bulky fluorescent derivatives of adenosine to the low affinity E2ATP site leads to inhibition of Na+/K+-ATPase. Analysis of structural requirements of fluorescent ATP derivatives with a Koshland-Némethy-Filmer model of two interacting ATP sites.
AuthorsThoenges D, Amler E, Eckert T, Schoner W
JournalJ Biol Chem
PubMed ID9890953
A Koshland-Némethy-Filmer model of two cooperating ATP sites has previously been shown to explain the kinetics of inhibition of Na+/K+-ATPase (EC 3.6.1.37) by dansylated ATP (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321). The present work demonstrates that this model adequately describes all types of interactions and ... More
Kinetics and motility of the Eg5 microtubule motor.
AuthorsLockhart A, Cross RA
JournalBiochemistry
PubMed ID8652578
We have investigated the kinetic properties of the slow plus end directed microtubule (MT) motor Eg5. The recombinantly expressed fusion protein E437GST, containing residues 12-437 of Eg5 fused to the N-terminus of glutathione S-transferase (GST), is dimeric and motile, translocating MTs at an average speed of 0.063 (+/-0.01) micrometers(-1). The ... More
Equilibrium and kinetic studies of substrate binding to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli.
AuthorsBermingham A, Bottomley JR, Primrose WU, Derrick JP
JournalJ Biol Chem
PubMed ID10751386
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (HMDP) by ATP to form 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, an intermediate in the pathway for folic acid biosynthesis. The enzyme has been identified as a potential target for antimicrobial drugs. Equilibrium binding studies showed that Escherichia coli HPPK-bound ATP or the nonhydrolyzable ATP analogue ... More
Pathway of ATP hydrolysis by monomeric and dimeric kinesin.
AuthorsMoyer ML, Gilbert SP, Johnson KA
JournalBiochemistry
PubMed ID9454569
The ATPase mechanism for a monomeric Drosophila kinesin construct, K341, was determined by pre-steady-state kinetic methods and compared to dimeric kinesin, K401. We directly measured the kinetics of binding mantATP (a fluorescent ATP analog) to the microtubule K341 complex, the dissociation of K341 from the microtubule, and release of phosphate ... More
Structural basis for the inhibition of mammalian membrane adenylyl cyclase by 2 '(3')-O-(N-Methylanthraniloyl)-guanosine 5 '-triphosphate.
AuthorsMou TC, Gille A, Fancy DA, Seifert R, Sprang SR
JournalJ Biol Chem
PubMed ID15591060
Membrane-bound mammalian adenylyl cyclase (mAC) catalyzes the synthesis of intracellular cyclic AMP from ATP and is activated by stimulatory G protein alpha subunits (Galpha(s)) and by forskolin (FSK). mACs are inhibited with high potency by 2 '(3')-O-(N-methylanthraniloyl) (MANT)-substituted nucleotides. In this study, the crystal structures of the complex between Galpha(s).GTPgammaS ... More
Binding of ATP to the fructose-2,6-bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase leads to activation of its 6-phosphofructo-2-kinase.
AuthorsYang QH, Zhu Z, Dong MQ, Ling S, Wu CL, Li L
JournalJ Biol Chem
PubMed ID11325970
To understand the mechanism by which the activity of the 6-phosphofructo-2-kinase (6PF-2K) of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is stimulated by its substrate ATP, we studied two mutants of the enzyme. Mutation of either Arg-279, the penultimate basic residue within the Walker A nucleotide-binding fold in the bisphosphatase domain, or Arg-359 to ... More
Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants: implications for the open-closed transition identified by crystallography.
AuthorsMálnási-Csizmadia A, Woolley RJ, Bagshaw CR
JournalBiochemistry
PubMed ID11123942
When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the converter region, responds to two independent conformational events on ... More
Differences in nucleotide-binding site of isoapyrases deduced from tryptophan fluorescence.
AuthorsEspinosa V, Kettlun AM, Zanocco A, Cardemil E, Valenzuela MA
JournalPhytochemistry
PubMed ID12657291
Comparative studies of intrinsic and extrinsic fluorescence of apyrases purified from two potato tuber varieties (Pimpernel and Desirée) were performed to determine differences in the microenvironment of the nucleotide binding site. The dissociation constants (K(d)) of Pimpernel apyrase for the binding of different fluorescent substrate analogs: methylanthranoyl (MANT-), trinitrophenyl (TNP-), ... More
Turnover of fluorescent nucleoside triphosphates by isolated immobilized myosin filaments. Transient kinetics on the zeptomole scale.
AuthorsSowerby AJ, Seehra CK, Lee M, Bagshaw CR
JournalJ Mol Biol
PubMed ID8230191
Recent developments of in vitro motility assays have allowed the sliding velocity and force generation to be measured when a single actin filament interacts with a small number of immobilized myosin molecules. In contrast, the associated ATPase activities have been estimated from the whole population of molecules in the flow ... More
Mechanism of microtubule kinesin ATPase.
AuthorsMa YZ, Taylor EW
JournalBiochemistry
PubMed ID7548088
A six-step mechanism is derived for the activation of kinesin K379 ATPase by microtubules. The data are fitted by the kinetic scheme [Formula see text] where T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; MtK refers to the complex of a K379 unit with ... More
Chemical mechanism of the fructose-6-phosphate,2-kinase reaction from the pH dependence of kinetic parameters of site-directed mutants of active site basic residues.
AuthorsMizuguchi H, Cook PF, Hasemann CA, Uyeda K
JournalBiochemistry
PubMed ID9220964
A bifunctional enzyme, fructose-6-phosphate 2-kinase-fructose 2, 6-bisphosphatase, catalyzes synthesis and degradation of fructose 2, 6-bisphosphate. Mutants of basic residues, including Lys51, Arg78, Arg79, Arg136, Lys172, and Arg193, immediately around the active site of rat testis fructose 6-P,2-kinase were constructed, and their steady state kinetics, ATP binding, and the effect of ... More
Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase.
AuthorsPechatnikova E, Taylor EW
JournalJ Biol Chem
PubMed ID9388211
The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, ... More
Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 1. Use of fluorescent nucleotide analogues.
AuthorsMoore KJ, Lohman TM
JournalBiochemistry
PubMed ID7981217
The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in a reaction that is coupled to ATP binding and hydrolysis. The Rep protein is a stable monomer in the absence of DNA but dimerizes upon binding either single-stranded or duplex DNA, and the dimer appears to be the ... More
Probing the nucleotide binding sites of axonemal dynein with the fluorescent nucleotide analogue 2'(3')-O-(-N-Methylanthraniloyl)-adenosine 5'-triphosphate.
AuthorsMocz G, Helms MK, Jameson DM, Gibbons IR
JournalBiochemistry
PubMed ID9657700
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 approximately 0.38) to dynein and can be displaced by ATP and ADP added to ... More
The mechanism of smooth muscle caldesmon-tropomyosin inhibition of the elementary steps of the actomyosin ATPase.
AuthorsAlahyan M, Webb MR, Marston SB, El-Mezgueldi M
JournalJ Biol Chem
PubMed ID16540476
Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin ... More
5'-(p-fluorosulfonylbenzoyl)-2'(or 3')-(methylanthraniloyl)adenosine, fluorescent affinity labels for adenine nucleotide binding sites: interaction with the kinase active site of the receptor for epidermal growth factor.
AuthorsScoggins RM, Summerfield AE, Stein RA, Guyer CA, Staros JV
JournalBiochemistry
PubMed ID8703925
We have found that the epidermal growth factor (EGF) receptor kinase can utilize the fluorescent ATP derivative, methylanthraniloyl ATP, as a substrate. On the basis of this observation, together with our previous studies that showed that 5'-(p-fluorosulfonylbenzoyl)adenosine (5'-FSBAdo) is a highly specific affinity label for the ATP site of the ... More
Study of the substrate-binding properties of bovine liver adenosine kinase and inhibition by fluorescent nucleoside analogues.
AuthorsPelicano H, Maury G, Elalaoui A, Shafiee M, Imbach JL, Goody RS, Divita G
JournalEur J Biochem
PubMed ID9342249
Adenosine kinase (AK) catalyzes the phosphorylation of adenosine to AMP with ATP as phosphate donor. Intrinsic fluorescence of bovine liver AK was shown previously to be a sensitive probe to quantify the binding of substrates to the enzyme [Elaloui, A., Divita, G., Maury, G., Imbach, J.-L. & Goody, R. S. ... More
Alternating site mechanism of the kinesin ATPase.
AuthorsGilbert SP, Moyer ML, Johnson KA
JournalBiochemistry
PubMed ID9454568
The processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results ... More
ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility.
AuthorsShimizu T, Thorn KS, Ruby A, Vale RD
JournalBiochemistry
PubMed ID10819995
Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed ... More
Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate.
AuthorsWhite HD, Belknap B, Webb MR
JournalBiochemistry
PubMed ID9305974
We have measured the kinetics of inorganic phosphate (Pi) release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myosin-ADP-Pi to actin. Myosin subfragment 1 and a series of nucleoside triphosphates were ... More
The active sites of fructose 6-phosphate,2-kinase: fructose-2, 6-bisphosphatase from rat testis. Roles of Asp-128, Thr-52, Thr-130, Asn-73, and Tyr-197.
AuthorsUyeda K, Wang XL, Mizuguchi H, Li Y, Nguyen C, Hasemann CA
JournalJ Biol Chem
PubMed ID9065453
To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate, 2-kinase:fructose-2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr-197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in ... More
Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides.
AuthorsGille A, Lushington GH, Mou TC, Doughty MB, Johnson RA, Seifert R
JournalJ Biol Chem
PubMed ID14981084
Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in ... More
Nup159 Weakens Gle1 Binding to Dbp5 But Does Not Accelerate ADP Release.
Authors
JournalJ Mol Biol
PubMed ID29782832
Heat Shock Factor 1 Is a Direct Antagonist of AMP-Activated Protein Kinase.