The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Nigel S Scrutton - One of the best experts on this subject based on the ideXlab platform.
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driving force analysis of proton tunnelling across a reactivity series for an Enzyme Substrate Complex
ChemBioChem, 2008Co-Authors: Parvinder Hothi, Anna Roujeinikova, Michael J Sutcliffe, David Leys, Paul M Cullis, Nigel S ScruttonAbstract:Quantitative structure-activity relationships are widely used to probe C-H bond breakage by quinoprotein Enzymes. However, we showed recently that p-substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the Enzyme-Substrate Complex prior to C-H bond breakage. This rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p-substituted benzylamines. Here we report reactivity (driving force) studies of AADH with p-substituted phenylethylamines for which the kinetic isotope effect (approximately 16) accompanying C-H/C-(2)H bond breakage is elevated above the semi-classical limit. We show bond breakage occurs by quantum tunnelling and that within the context of the environmentally coupled framework for H-tunnelling the presence of the p-substituent places greater demand on the apparent need for fast promoting motions. The crystal structure of AADH soaked with phenylethylamine or methoxyphenylethylamine indicates that the structural change identified with p-substituted benzylamines should not limit the reaction with p-substituted phenylethylamines. This is consistent with the elevated kinetic isotope effects measured with p-substituted phenylethylamines. We find a good correlation in the rate constant for proton transfer with bond dissociation energy for the reactive C-H bond, consistent with a rate that is limited by a Marcus-like tunnelling mechanism. As the driving force becomes larger, the rate of proton transfer increases while the Marcus activation energy becomes smaller. This is the first experimental report of the driving force perturbation of H-tunnelling in Enzymes using a series of related Substrates. Our study provides further support for proton tunnelling in AADH.
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conformational events during ternary Enzyme Substrate Complex formation are rate limiting in the catalytic cycle of the light driven Enzyme protochlorophyllide oxidoreductase
Biochemistry, 2008Co-Authors: Derren J Heyes, Binuraj R K Menon, Michiyo Sakuma, Nigel S ScruttonAbstract:The light-driven Enzyme, protochlorophyllide oxidoreductase (POR), has proven to be an excellent model system for studying the role of protein motions during catalysis. POR catalyzes the trans addition of hydrogen across the C17-C18 double bond of protochlorophyllide (Pchlide), which is a key step in chlorophyll biosynthesis. While we currently have a detailed understanding of the initial photochemical events and the subsequent hydrogen transfer reactions, there remains a lack of information about the slower Substrate binding events leading to the formation of the catalytically active ternary Complex. As POR is light-activated, it is relatively straightforward to isolate the ternary Enzyme-Substrate Complex in the dark prior to catalysis, which has facilitated the use of a variety of spectroscopic and kinetic probes to study the binding of both Substrates. Herein, we provide a detailed kinetic and thermodynamic description of these processes and show that the binding events are Complex, involving multiple conformational states en route to the formation of a ternary Complex that is primed for photoactivation. The initial binding of NADPH involves three distinct steps, which appear to be necessary for the optimal alignment of the cofactor in the Enzyme active site. This is followed by the binding of the Pchlide Substrate and subsequent Substrate-induced conformational changes within the Enzyme that occur prior to the formation of the final "poised" conformational state. These studies, which provide important information on the formation of the reactive conformation, reveal that ternary Complex formation is the rate-limiting step in the overall reaction and is controlled by slow conformational changes in the protein.
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the ph dependence of kinetic isotope effects in monoamine oxidase a indicates stabilization of the neutral amine in the Enzyme Substrate Complex
FEBS Journal, 2008Co-Authors: Rachel V Dunn, Ker R Marshall, Andrew W Munro, Nigel S ScruttonAbstract:A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine Substrate in the Enzyme–Substrate Complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine Substrate that binds to the Enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated Substrates. For all Substrates used, there is a macroscopic ionization in the Enzyme–Substrate Complex attributed to a deprotonation event required for optimal catalysis with a pKa of 7.4–8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine Substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the Substrate binding constant (kred/Ks, reporting on ionizations in the free Enzyme and/or free Substrate) is due to deprotonation of the free Substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the Enzyme at high pH. The pKa of the free amine is above 9.3 for all Substrates, and is greatly perturbed (ΔpKa∼ 2) on binding to the Enzyme active site. This perturbation of the Substrate amine pKa on binding to the Enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the Substrate in the Enzyme–Substrate Complex, thus enabling efficient functioning of these Enzymes at physiologically relevant pH.
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reductive half reaction of the h172q mutant of trimethylamine dehydrogenase evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the Enzyme Substrate Complex
Biochemical Journal, 1999Co-Authors: Jaswir Basran, Michael J Sutcliffe, Russ Hille, Nigel S ScruttonAbstract:The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172-->Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the Substrate in a 'carbanion' mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type Enzyme with perdeuterated trimethylamine as Substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the Enzyme-Substrate Complex, with macroscopic pKa values of 6.5+/-0.2 and 8.4+/-0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4+/-0.1 and 10.5+/-0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4+/-0.1 for the wild-type Enzyme-Substrate Complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type Enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on Substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the Substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.
Florian Stengel - One of the best experts on this subject based on the ideXlab platform.
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structural dynamics of the e6ap ube3a e6 p53 Enzyme Substrate Complex
Nature Communications, 2018Co-Authors: Carolin Sailer, Fabian Offensperger, Alexandra Julier, Kaimichael Kammer, Ryan Walkergray, Matthew G Gold, Martin Scheffner, Florian StengelAbstract:Deregulation of the ubiquitin ligase E6AP is causally linked to the development of human disease, including cervical cancer. In Complex with the E6 oncoprotein of human papillomaviruses, E6AP targets the tumor suppressor p53 for degradation, thereby contributing to carcinogenesis. Moreover, E6 acts as a potent activator of E6AP by a yet unknown mechanism. However, structural information explaining how the E6AP-E6-p53 Enzyme-Substrate Complex is assembled, and how E6 stimulates E6AP, is largely missing. Here, we develop and apply different crosslinking mass spectrometry-based approaches to study the E6AP-E6-p53 interplay. We show that binding of E6 induces conformational rearrangements in E6AP, thereby positioning E6 and p53 in the immediate vicinity of the catalytic center of E6AP. Our data provide structural and functional insights into the dynamics of the full-length E6AP-E6-p53 Enzyme-Substrate Complex, demonstrating how E6 can stimulate the ubiquitin ligase activity of E6AP while facilitating ubiquitin transfer from E6AP onto p53.
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Structural dynamics of the E6AP/UBE3A-E6-p53 Enzyme-Substrate Complex.
Nature Communications, 2018Co-Authors: Carolin Sailer, Fabian Offensperger, Alexandra Julier, Kaimichael Kammer, Matthew G Gold, Martin Scheffner, Ryan Walker-gray, Florian StengelAbstract:Deregulation of the ubiquitin ligase E6AP is causally linked to the development of human disease, including cervical cancer. In Complex with the E6 oncoprotein of human papillomaviruses, E6AP targets the tumor suppressor p53 for degradation, thereby contributing to carcinogenesis. Moreover, E6 acts as a potent activator of E6AP by a yet unknown mechanism. However, structural information explaining how the E6AP-E6-p53 Enzyme-Substrate Complex is assembled, and how E6 stimulates E6AP, is largely missing. Here, we develop and apply different crosslinking mass spectrometry-based approaches to study the E6AP-E6-p53 interplay. We show that binding of E6 induces conformational rearrangements in E6AP, thereby positioning E6 and p53 in the immediate vicinity of the catalytic center of E6AP. Our data provide structural and functional insights into the dynamics of the full-length E6AP-E6-p53 Enzyme-Substrate Complex, demonstrating how E6 can stimulate the ubiquitin ligase activity of E6AP while facilitating ubiquitin transfer from E6AP onto p53.
John J Holbrook - One of the best experts on this subject based on the ideXlab platform.
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source of catalysis in the lactate dehydrogenase system ground state interactions in the Enzyme Substrate Complex
Biochemistry, 1994Co-Authors: Hua Deng, Jie Zheng, Anthony Clarke, John J HolbrookAbstract:: The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these Enzymes. The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when Complexed to these Enzymes, which shows that the group remains ionized in the ion pair Complex with Arg-171 on the Enzyme. The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm-1 to a lower frequency than observed for the carbonyl of unliganded pyruvate in the bacterial Enzyme because of polarization of the carbonyl bond. Thus, the bacterial Enzyme shows the same Substrate activation because of the C(+)-O- charge separation that was seen previously with the mammalian Enzymes. On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the Enzyme and the adduct C = O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C V O and the protonated His-195. Thus, while the C = O bond is polarized on the Enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stability substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary Complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Yuri F Zuev - One of the best experts on this subject based on the ideXlab platform.
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Brownian dynamics simulation of Substrate motion near active site of Enzyme entrapped inside reverse micelle.
European Biophysics Journal, 2010Co-Authors: Elena A Ermakova, Natalia L Zakhartchenko, Yuri F ZuevAbstract:Brownian dynamics simulation has been applied to analyze the influence of the electrostatic field of a reverse micelle on the Enzyme-Substrate Complex formation inside a micelle. The probability that the Enzyme-Substrate Complex will form from serine protease (trypsin) and the specific hydrophilic cationic Substrate Nα-benzoyl-l-arginine ethyl ester has been studied within the framework of the encounter Complex formation theory. It has been shown that surfactant charge, dipole moments created by charged surfactant molecules and counterions, and permittivity of the inner core of reverse micelles can all be used as regulatory parameters to alter the Substrate orientation near the active site of the Enzyme and to change the probability that the Enzyme-Substrate Complex will form.
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effect of surface potential of reverse micelle on Enzyme Substrate Complex formation
Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2008Co-Authors: Elena A Ermakova, Natalia L Zakhartchenko, Yuri F ZuevAbstract:Abstract The influence of the electrostatic potential of reverse micelle on the Enzyme–Substrate Complex formation have been studied within the framework of the encounter Complex (EC) formation theory. Reverse micelles have a multiple-factor effect on encapsulated substances and on mechanisms of their interaction. The action of individual constituents on the overall micellar effect was analyzed by means of Brownian dynamics simulation. The effects of surface potential in charged and neutral reverse micelles, of the size and of the ionic strength of micellar core on the probability to form the reaction Complex between serine protease (trypsin) and specific Substrate Nα-benzoyl- l -arginine ethyl ester was examined. It was shown that negative potential of micelle increases the probability of EC formation and the positive potential decreases it. Orientation of Substrate in electrostatic field of micelle depends on the value and the sign of surface potential.
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Effect of surface potential of reverse micelle on Enzyme–Substrate Complex formation
Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2008Co-Authors: Elena A Ermakova, Natalia L Zakhartchenko, Yuri F ZuevAbstract:Abstract The influence of the electrostatic potential of reverse micelle on the Enzyme–Substrate Complex formation have been studied within the framework of the encounter Complex (EC) formation theory. Reverse micelles have a multiple-factor effect on encapsulated substances and on mechanisms of their interaction. The action of individual constituents on the overall micellar effect was analyzed by means of Brownian dynamics simulation. The effects of surface potential in charged and neutral reverse micelles, of the size and of the ionic strength of micellar core on the probability to form the reaction Complex between serine protease (trypsin) and specific Substrate Nα-benzoyl- l -arginine ethyl ester was examined. It was shown that negative potential of micelle increases the probability of EC formation and the positive potential decreases it. Orientation of Substrate in electrostatic field of micelle depends on the value and the sign of surface potential.
Jeffrey A. Haas - One of the best experts on this subject based on the ideXlab platform.
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Fluorescence anisotropy studies of Enzyme-Substrate Complex formation in stearoyl-ACP desaturase.
Biochemistry, 2002Co-Authors: Jeffrey A. HaasAbstract:: Stearoyl-acyl carrier protein Delta(9)-desaturase (delta9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e(-) reduced delta9D. Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting delta9D was observed with K(D1) = 13 +/- 3 nM. No significant difference in the K(D1) value was observed for 4e(-) delta9D. An approximately 4-fold increase in K(D1) per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0. In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting delta9D was estimated to have K(D2) approximately 350 +/- 40 nM. The K(D2) values exhibited a similar dependence on acyl chain length as observed for the K(D1) values. The k(off) values measured by stopped-flow anisotropy measurements for reversal of the Enzyme-Substrate Complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s(-)(1)) relative to 18:0-ACP (1 s(-)(1)). Increases in acyl chain length are thus associated with the presently reported increases in the K(D) and k(off) values. These results indicate that acyl chain length selectivity derives in major part from partition of the Enzyme-Substrate Complex between Substrate release and subsequent steps in catalysis.
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fluorescence anisotropy studies of Enzyme Substrate Complex formation in stearoyl acp desaturase
Biochemistry, 2002Co-Authors: Jeffrey A. HaasAbstract:Stearoyl-acyl carrier protein Δ 9-desaturase (Δ9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow...