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M R Webb

Publications and source records attributed to M R Webb.

At least 55 records · Page 3Linked to original sources
PubMed1991

Fluorescence approaches to the study of the p21ras GTPase mechanism.

The use of ribose-modified guanine nucleotides and tryptophan mutants of p21ras, neither of which have significant effect on the kinetic mechanism of the p21ras GTPase and the GAP-activated p21ras GTPase, will now allow a detailed kinetic study of how GAP and other regulatory proteins interact with p21ras. This will lead to a better understanding of how the relative concentrations of 'active' p21ras. GTP and 'inactive' p21ras. GDP are regulated in the cell.

GTP Phosphohydrolases↗
PubMed1990

Kinetics of ATP and inorganic phosphate release during hydrolysis of ATP by rabbit skeletal actomyosin subfragment 1. Oxygen exchange between water and ATP or phosphate.

We have used the technique of phosphate: water oxygen exchange to measure the rate of ATP and Pi release and Pi binding to myosin subfragment 1 and actomyosin subfragment 1 from rabbit skeletal muscle. The oxygen exchange distributions for ATP and Pi release fit a simple kinetic model with a single set of rate constants for each step. For actomyosin subfragment 1 (20 degrees C, pH 7.0, I = 50 mM), the rate constant governing ATP release is approximately 8 s-1, Pi release is at approximately 60 s-1 and Pi rebinds to an ADP state at greater than 120 M-1 s-1. These rate constants are similar to those that may occur for undistorted cross-bridges within glycerinated rabbit psoas fibers (Bowater, R., Webb, M. R., and Ferenczi, M. A. (1989) J. Biol. Chem. 264, 7193-7201.

Actins↗
PubMed1990

Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site.

2'(3')-O-(N-Methyl)anthraniloylguanosine 5'-triphosphate (mantGTP) is a fluorescent analogue of GTP that has similar properties to the physiological substrate in terms of its binding constant and the kinetics of its interactions with p21NRAS, the NRAS protooncogene product. There is a 3-fold increase in fluorescence intensity when mantGTP binds to p21NRAS. The rate constant for the cleavage of mantGTP complexed with the protein is similar to that of GTP, and cleavage is accompanied by a fluorescence intensity change in the wild-type protein complex. A two-phase fluorescence change also occurs when the nonhydrolyzable analogue 2'(3')-O-(N-methyl)anthraniloylguanosine 5'-[beta, gamma-imido]triphosphate (mantp[NH]ppG) binds to wild-type p21NRAS. The second phase occurs at the same rate as the second phase observed after mantGTP binding. Thus this second phase is probably a conformation change of the p21NRAS nucleotiside triphosphate complex and that the change controls the rate of GTP hydrolysis on the protein. With a transforming mutant, [Asp12]-p21NRAS, there is no second phase of the fluorescence change after mantGTP or mantp[NH]ppG binding, even though mantGTP is hydrolyzed. This shows that an equivalent conformational change does not occur and thus the mutant may stay in a "GTP-like" conformation throughout the GTPase cycle. These results are discussed in terms of the proposed role of p21NRAS in signal transduction and the transforming properties of the mutant.

Binding Sites↗
PubMed1989

Measurement of the reversibility of ATP binding to myosin in calcium-activated skinned fibers from rabbit skeletal muscle. Oxygen exchange between water and ATP released to the solution.

We have measured the rate constant for ATP release from myosin heads of Ca2+-activated, demembranated muscle fibers using the technique of phosphate-water oxygen exchange. Single rabbit psoas fibers were held in an activating solution in [18O]water ([MgATP] = 8 mM, ionic strength = 0.2 M, pH = 7.0, 24 degrees C). After about 20% hydrolysis of ATP, product Pi and remaining ATP were isolated, and the distribution of 18O in both molecules was analyzed using a mass spectrometer. The exchange in Pi was similar to that previously reported (Hibberd, M. G., Webb, M. R., Goldman, Y. E., and Trentham, D. R. (1985) J. Biol. Chem. 260, 3496-3501). The amount of 18O in ATP gave a rate constant of about 4 s-1 for ATP release, if it is assumed that each rate constant in the pathway of ATP hydrolysis has the same value for all myosin ATPase sites. However, the distribution of 18O in both released Pi and ATP is not well explained by a single pathway for ATP hydrolysis. We present a model that indicates how such distributions could arise from a range of values for the rate constants for Pi and ATP release from actomyosin, and this range is determined by differences in the amounts of strain in attached crossbridges. The kinetic information obtained from these isotope exchange experiments is compared to show that they give a compatible set of rate constants for actomyosin in fibers.

Actomyosin↗
PubMed1989

The mechanism of guanosine nucleotide hydrolysis by p21 c-Ha-ras. The stereochemical course of the GTPase reaction.

The use of guanosine 5'-O-(gamma-thio)triphosphate as a substrate for p21 c-Ha-ras was established. By using chirally labeled [gamma-17O,18O]guanosine 5'-O-(gamma-thio)triphosphate, the stereochemical course of the GTPase reaction was determined. The analysis shows that the hydrolysis occurs with inversion at the gamma-phosphorus. This shows that the most likely mechanism is a single step, in-line transfer, without a phosphoenzyme or other phosphorylated intermediate.

Adenosine Triphosphate↗
PubMed1988

Kinetic analysis of the hydrolysis of GTP by p21N-ras. The basal GTPase mechanism.

The rate constants have been determined for elementary steps in the basal GTPase mechanism of normal p21N-ras (Gly-12) and an oncogenic mutant (Asp-12): namely GTP binding, hydrolysis, phosphate release, and GDP release. By extrapolation from data at lower temperatures, the GTP association rate constant at 37 degrees C is 1.4 x 10(8) M-1 s-1 for the normal protein and 4.8 x 10(8) M-1 s-1 for the mutant. Other rate constants were measured directly at 37 degrees C, and three processes have similar slow values. GTP dissociation is at 1.0 x 10(-4) s-1 (normal) and 5.0 x 10(-4) s-1 (mutant). The hydrolysis step is at 3.4 x 10(-4) s-1 (normal) and 1.5 x 10(-4) s-1 (mutant). GDP dissociates at 4.2 x 10(-4) s-1 (normal) and 2.0 x 10(-4) s-1 (mutant). GDP association rate constants are similar to those for GTP, 0.5 x 10(8) M-1 s-1 for normal and 0.7 x 10(8) M-1 s-1 for mutant. Both hydrolysis and GDP release therefore contribute to rate limitation of the basal GTPase activity. There are distinct differences (up to 5-fold) between rate constants for the normal and mutant proteins at a number of steps. The values are consistent with the reduced GTPase activity for this mutant and suggest little difference between normal and mutant proteins in the relative steady-state concentrations of GTP and GDP complexes that may represent active and inactive states. The results are discussed in terms of the likely role of p21ras in transmembrane signalling.

Escherichia coli↗
PubMed1988

Changes in the ATPase activity of insect fibrillar flight muscle during sinusoidal length oscillation probed by phosphate-water oxygen exchange.

Extensive phosphate-water oxygen exchange occurs when ATP is hydrolyzed in an [18O]water medium by length oscillated and Ca2+-activated, chemically skinned fibers from the flight muscle of the giant waterbug Lethocerus indicus. For fibers which are length oscillated under conditions not optimal for ATPase activity or oscillatory work, the pattern of exchange shows two pathways for hydrolysis. One pathway has low exchange, because steps controlling Pi release are rapid; the other pathway has high exchange and slow Pi release. Steps controlling Pi release appear rate-limiting for changes in the high-exchange ATPase activity that occur on varying the frequency and amplitude of oscillation. On length oscillation under conditions of optimal ATPase activity or work, only the high-exchange pathway is present. Cross-bridges following the high-exchange pathway are therefore responsible for oscillatory work, the physiological function of the muscle, and behave uniformly with respect to oxygen exchange. The single pathway and the magnitude of the ATPase activity are both similar to results with isometric strained fibers (Lund, J., Webb, M. R., and White, D. C. S. (1987) J. Biol. Chem. 262, 8584-8590). A qualitative model is suggested for oscillatory work by cross-bridges, arising from the common periodicity of the thick and thin filaments in insect flight muscle.

Adenosine Triphosphatases↗
PubMed1988

Cross-bridge kinetics in asynchronous insect flight muscle.

Calcium-activated insect flight muscle is further activated by small applied strain. We have investigated the mechanism of strain activation by measuring the extent of oxygen exchange between phosphate and water during ATP hydrolysis by Ca2+ -activated, chemically skinned fibers from the flight muscle of the giant waterbug Lethocerus indicus at different degrees of strain. The maximally activated insect fibres show a pattern of oxygen exchange which is well fitted by a single pathway of ATP hydrolysis. This differs from results with rabbit muscle, which show a more complicated form of oxygen exchange. We ascribe this difference to the different pattern of distribution of myosin heads on the thick filament. Calcium-activated, but unstrained, insect fibres also require more than a simple pathway of ATP hydrolysis to account for the pattern of oxygen exchange. The transition between the more complex pathway and the simple pathway occurs over a very narrow range of strain, centred at zero strain (rest length in the relaxed fibres). We describe how the experimental results of strain activation might occur, based upon Wray's description of filament geometries, but taking into account likely sarcomere mechanics. The pattern of oxygen exchange found in fully activated insect fibres suggests that the process of phosphate release from the actomyosin is rate limiting, implying that the main tension-generating state is an AM.ADP.Pi complex.

Actomyosin↗
PubMed1987

Changes in the ATPase activity of insect fibrillar flight muscle during calcium and strain activation probed by phosphate-water oxygen exchange.

During ATP hydrolysis by Ca2+-activated chemically skinned fibers from the flight muscle of the giant waterbug Lethocerus indicus, there is extensive phosphate-water oxygen exchange. For unstrained fibers the pattern of exchange shows that there is more than one pathway for hydrolysis, due to the ATPase activity of cross-bridges. Multiple pathways are an established property of both vertebrate actomyosin and fibers. The pattern of exchange can be fitted by two pathways: one with low exchange because the step(s) controlling Pi release are rapid, the other with high exchange and slow Pi release. The high-exchange pathway is responsible for most of the increase in ATPase activity on Ca2+ activation. On strain activation, only the high-exchange pathway is present, accounting for all the ATPase increase and responsible for force generation. In fully activated fibers, the cross-bridges which hydrolyze ATP and generate force behave uniformly with respect to oxygen exchange. The exchange pattern shows that the rate of Pi release changes dramatically over a very narrow strain increase. Step(s) controlling Pi release are at least partially rate-limiting for the overall ATPase reaction. The results are discussed in relation to models for strain activation and the identity of force-generating states.

Adenosine Triphosphatases↗
PubMed1987

Analysis of the ATPase mechanism of myosin subfragment 1 from insect fibrillar flight muscle in the presence and absence of actin, using phosphate-water oxygen exchange measurements.

Phosphate-water oxygen exchange was measured during ATPase activity of myosin subfragment 1, isolated from Lethocerus flight muscle. The result supports a mechanism including a rapid reversible ATP cleavage step followed by slow Pi release, so that extensive exchange occurs by multiple reversals of the cleavage step. Interaction with actin accelerates Pi release and reduces the extent of exchange. These properties are similar to vertebrate skeletal muscle subfragment 1. Differences in kinetic properties between insect flight and rabbit skeletal muscle exhibited in the fibres, particularly in respect of the strain activation of insect flight muscle, are not exhibited in the isolated myosin heads and are therefore probably due to organization within the fibre lattice.

Actins↗
PubMed1986

Absence of a phosphorylated intermediate during ATP hydrolysis by Escherichia coli transcription termination protein rho.

We have established the suitability of adenosine 5'-O-(gamma-thio)triphosphate(ATP gamma S) as an analog of ATP for the nucleoside triphosphatase activity of Escherichia coli transcription termination protein rho (EC 3.6.1.3). Steady-state analysis gives a Vmax of 1.5 mumol min-1 mg-1, 9% of the value with MgATP as substrate, and indicates that ATP gamma S binds as tightly (based on Km and Ki versus ATP) to rho as does ATP. (gamma-S)[beta gamma-17O,gamma-17O,gamma-18O]ATP gamma S was used as substrate to produce chiral product inorganic [17O,18O]thiophosphate and determine the stereochemical course of the hydrolysis. The results of this determination, inversion at the thiophosphoryl phosphorus, indicate that the enzymatic hydrolysis of ATP by rho consists of a direct transfer of the phospho group to water without the existence of a phosphoenzyme or phospho-RNA intermediate.

Adenosine Triphosphate↗
PubMed1986

Oxygen exchange between Pi in the medium and water during ATP hydrolysis mediated by skinned fibers from rabbit skeletal muscle. Evidence for Pi binding to a force-generating state.

Oxygen exchange between (18O4)Pi in the medium and water accompanies ATP hydrolysis catalyzed by the calcium-regulated MgATPase of vertebrate skeletal muscle. Exchange was observed in chemically skinned fibers from rabbit psoas muscle held isometrically and activated by 30 microM free Ca2+. The rate of exchange was approximately proportional to Pi concentration (up to 10 mM) and was characterized by an apparent second order rate constant greater than or equal to 475 M-1 S-1 (pH 7.1, ionic strength 0.2 M, 22 degrees C). Much less exchange occurred in the absence of Ca2+ or when ATP was replaced by ADP. It has been inferred from mechanical experiments that Pi can bind to a force-generating ADP-bound state of actomyosin with resultant suppression of force (Hibberd, M. G., Dantzig, J. A., Trentham, D. R., and Goldman, Y. E. (1985) Science 228, 1317-1319). The oxygen exchange results support this inference by providing direct evidence that Pi in the medium binds at the ATPase catalytic site in activated isometric fibers. The inter-relationship of these two effects involving Pi on mechanochemical coupling in muscle is discussed.

Adenosine Diphosphate↗
PubMed1985

Oxygen exchange between phosphate and water accompanies calcium-regulated ATPase activity of skinned fibers from rabbit skeletal muscle.

The extent of oxygen exchange between phosphate and water has been measured for the calcium-regulated magnesium-dependent ATPase activity of chemically skinned fibers from rabbit skeletal muscle. The oxygen exchange was determined for isometrically held fibers by measuring with a mass spectrometer the distribution of 18O atoms in the product inorganic phosphate when ATP hydrolysis was carried out in H2(18)O. The extent of exchange was much greater in relaxed muscle (free Ca2+ less than 10(-8) M) than in calcium-activated muscle (free Ca2+ approximately equal to 3 X 10(-5) M). Activated fibers had an ATPase activity at least 30-fold greater than the relaxed fibers. These results correlate well with the extents of oxygen exchange accompanying magnesium-dependent myosin and unregulated actomyosin ATPase activities, respectively. In relaxed fibers, comparison of the amount of exchange with the ATPase activity suggests that the rate constant for the reformation of myosin-bound ATP from the myosin products complex is about 10 s-1 at 20 degrees C and pH 7.1. In each experiment the distribution of 18O in the Pi formed was incompatible with a single pathway for ATP hydrolysis. In the case of the calcium-activated fibers, the multiple pathways for ATP hydrolysis appeared to be an intrinsic property of the actomyosin ATPase in the fiber. These results indicate that in muscle fibers, as in isolated actomyosin, cleavage of protein-bound ATP is readily reversible and that association of the myosin products complex with actin promotes Pi release.

Animals↗
PubMed1984

The stereochemical course of phospho transfer catalyzed by adenylosuccinate synthetase. A reaction pathway via a phosphorylated intermediate with net inversion.

The stereochemical course of phospho transfer in the reaction catalyzed by adenylosuccinate synthetase from rat muscle has been determined with chiral [gamma-17O,18O]GTP gamma S as a substrate. The stereochemical configuration of the product, inorganic thiophosphate, was determined by 31P NMR after the compound was stereospecifically incorporated into ATP beta S. The reaction goes with net inversion of configuration, which is the course for a single phospho transfer, even though 6-phospho-IMP is probably an intermediate on the normal reaction pathway (Liebermann, I. (1956) J. Biol. Chem. 223, 327-339). The breakdown of this intermediate goes by C-O bond cleavage and so is not a true phospho transfer step. Thus, inversion of configuration during the course of this ligase reaction is consistent with a single phospho transfer step in the overall reaction, the formation of the phosphorylated intermediate.

Adenylosuccinate Synthase↗
PubMed1984

Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate.

In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87, 301-316]. We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O. These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.

Adenosine Triphosphate↗
PubMed1982

Characterization of the GTPase reaction of elongation factor Tu. Determination of the stereochemical course in the presence of antibiotic X5108.

The stereochemical course of the GTPase of elongation factor Tu from Escherichia coli has been determined by making use of the reaction dependent on antibiotic X5108 (an N-methylated derivative of kirromycin). Guanosine 5'-(gamma-thio)triphosphate stereospecifically labeled with 17O and 18O in the gamma-position was hydrolyzed in the presence of elongation factor Tu and X5108. The configuration of the product, inorganic [16O, 17O, 18O]thiophosphate was analyzed by 31P NMR after its stereospecific incorporation into adenosine 5'-(beta-thio)triphosphate. The analysis showed that the hydrolysis proceeds with inversion of configuration at the transferred phosphorus, implying that there is not a phosphoenzyme intermediate. Incubation of [beta gamma-18O, gamma-18O3]GTP with elongation factor Tu leaves the 18O-labeling unaltered, as shown by 31P NMR. No exchange of oxygens with water nor beta gamma-beta positional isotope exchange occurs, implying that not even transient cleavage can occur with the elongation factor alone. Only on interaction with X5108, kirromycin, or ribosomes does the cleavage occur, most likely by a single step, in-line transfer of the terminal phosphorus from GDP to a water oxygen. These properties of the GTP hydrolysis mechanism of elongation factor Tu are similar to those of elongation factor G.

Adenosine Triphosphate↗
PubMed1982

Electron paramagnetic resonance studies of MN(II) complexes with myosin subfragment 1 and oxygen 17-labeled ligands.

Ligands in the first coordination sphere of Mn(II) in the complex of MnADP with myosin subfragment 1 from rabbit skeletal muscle have been investigated. EPR spectroscopy was used to detect superhyperfine coupling between unpaired electrons of the metal ion and the nuclei of oxygen atoms specifically labeled with oxygen 17. The results show that ADP is a beta-monodentate ligand for Mn(II) and that there are probably two water oxygens directly bound to Mn(II). The inhibitory complex of vanadate with subfragment 1 . MnADP was also investigated. Vanadate-dependent changes in the EPR spectra for enzyme-bound Mn(II) indicate that the coordination sphere of MN(II) changes upon binding of vanadate. ADP remains a beta-monodenate ligand in the complex and experiments with 17O-labeled water indicate that two oxygen atoms originally in water are ligands in the complex. However, the oxygens of vanadate equilibrate with those of water during sample preparation so that one of these ligands may be a vanadate oxygen. Three additional ligands, probably from the protein, are required to complete the sextet of ligands to Mn(II) in both complexes studied.

Adenosine Diphosphate↗