Chromophoric spin-labeled β-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C β-lactamases

Mustafi, Devkumar; Hofer, Jennifer; Huang, Wanzhi; Palzkill, Timothy; Makinen, Marvin W.

doi:10.1016/j.saa.2003.10.024

Cited by 7 publications

(6 citation statements)

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“…Several organic solvents are commonly used in cryoenzymology studies because of their low viscosity and relatively low dielectric constant of mixtures with water . Among them are ethylene glycol, ,, methanol, , and DMSO. , However, ethylene glycol and methanol inactivate the EAL holoenzyme, , and ethylene glycol is too viscous for reproducible mixing at the low temperatures ( T ≤ 230 K) required to kinetically arrest the Co II −substrate radical pair formation reaction. Therefore, we chose DMSO as the cryosolvent.…”

Section: Methodsmentioning

confidence: 99%

Kinetic and Thermodynamic Characterization of Co^II−Substrate Radical Pair Formation in Coenzyme B₁₂-Dependent Ethanolamine Ammonia-Lyase in a Cryosolvent System by Using Time-Resolved, Full-Spectrum Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Wang

Warncke

2008

J. Am. Chem. Soc.

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The formation of the Co(II)-substrate radical pair catalytic intermediate in coenzyme B12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been studied by using time-resolved continuous-wave electron paramagnetic resonance (EPR) spectroscopy in a cryosolvent system. The 41% v/v DMSO/water cryosolvent allows mixing of holoenzyme and substrate, (S)-2-aminopropanol, at 230 K under conditions of kinetic arrest. Temperature step from 230 to 234-248 K initiates the cleavage of the cobalt-carbon bond and the monoexponential rise (rate constant, k(obs) = tau(obs)(-1)) of the EPR-detected Co(II)-substrate radical pair state. The detection deadtime: tau(obs) ratio is reduced by >10(2), relative to millisecond rapid mixing experiments at ambient temperatures. The EPR spectrum acquisition time is 5tau(obs), the approximately 10(2)-fold slower rate of the substrate radical rearrangement reaction relative to k(obs), and the reversible temperature dependence of the amplitude indicate that the Co(II)-substrate radical pair and ternary complex are essentially at equilibrium. The reaction is thus treated as a relaxation to equilibrium by using a linear two-step, three-state mechanism. The intermediate state in this mechanism, the Co(II)-5'-deoxyadenosyl radical pair, is not detected by EPR at signal-to-noise ratios of 10(3), which indicates that the free energy of the Co(II)-5'-deoxyadenosyl radical pair state is >3.3 kcal/mol, relative to the Co(II)-substrate radical pair. Van't Hoff analysis yields DeltaH13 = 10.8 +/- 0.8 kcal/mol and DeltaS13 = 45 +/- 3 cal/mol/K for the transition from the ternary complex to the Co(II)-substrate radical pair state. The free energy difference, DeltaG13, is zero to within one standard deviation over the temperature range 234-248 K. The extrapolated value of DeltaG13 at 298 K is -2.6 +/- 1.2 kcal/mol. The estimated EAL protein-associated contribution to the free energy difference is DeltaG(EAL) = -24 kcal/mol at 240 K, and DeltaH(EAL) = -13 kcal/mol and DeltaS(EAL) = 38 cal/mol/K. The results show that the EAL protein makes both strong enthalpic and entropic contributions to overcome the large, unfavorable cobalt-carbon bond dissociation energy, which biases the reaction in the forward direction of Co-C bond cleavage and Co(II)-substrate radical pair formation.

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Section: Methodsmentioning

confidence: 99%

Kinetic and Thermodynamic Characterization of Co^II−Substrate Radical Pair Formation in Coenzyme B₁₂-Dependent Ethanolamine Ammonia-Lyase in a Cryosolvent System by Using Time-Resolved, Full-Spectrum Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Wang

Warncke

2008

J. Am. Chem. Soc.

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“…The second approach is to slow the reaction, through manipulation of the Boltzmann thermal contribution, by lowering the temperature. This family of “cryoenzymology” methods (Fink, 1977; Travers & Barman, 1995) has been used to study a large selection of enzymes in the sub-273 K temperature range (Douzou, Sireix, & Travers, 1970; Feig, Ammons, & Uhlenbeck, 1998; Mustafi, Hofer, Huang, Palzkill, & Makinen, 2004; Travers, Bertrand, Roseau, & Vanthoai, 1978). The cryoenzymology systems are in the fluid (or supercooled fluid) state, and therefore, reaction can be initiated by mixing components ( e .…”

Section: Low-temperature Fluid Cryosolvent Systemmentioning

confidence: 99%

“…To achieve these criteria, several organic solvents can be used, owing to their high miscibility with water, low viscosity and relatively high dielectric constants(Douzou, 1977). Among them are ethylene glycol (Bicknell & Waley, 1985; Tesi, Travers, & Barman, 1990; Travers et al, 1978), methanol (Bicknell & Waley, 1985; Feig et al, 1998), propanediol (Fahy, Lilley, Linsdell, Douglas, & Meryman, 1990; Mehl, 1993), dimethyl sulfoxide (DMSO) (Douzou et al, 1970; Mustafi et al, 2004), and their ternary or higher-order mixtures (Bragger, Dunn, & Daniel, 2000). For the EAL enzyme, methanol and ethylene glycol react with, and inactivate, the holoenzyme (Babior, 1970; Babior & Krouwer, 1979).…”

Section: Low-temperature Fluid Cryosolvent Systemmentioning

confidence: 99%

Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems

Wang

Zhu

Kohne

et al. 2015

Methods in Enzymology

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Approaches to the resolution and characterization of individual chemical steps in enzyme catalytic sequences, by using temperatures in the cryogenic range of 190–250 K, and kinetics measured by time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy in fluid cryosolvent and frozen solution systems, are described. The preparation and performance of the adenosylcobalamin-dependent ethanolamine ammonia-lyase enzyme from Salmonella typhimurium in the two systems exemplifies the biochemical and spectroscopic methods. General advantages of low-temperature studies are: (1) Slowing of reaction steps, so that measurements can be made by using straightforward T-step kinetic methods and commercial instrumentation, (2) resolution of individual reaction steps, so that first-order kinetic analysis can be applied, and (3) accumulation of intermediates that are not detectable at room temperatures. The broad temperature range from room temperature to 190 K encompasses three regimes: (1) Temperature-independent mean free energy surface (corresponding to native behavior), (2) the narrow temperature region of a glass-like transition in the protein, over which the free energy surface changes, revealing dependence of the native reaction on collective protein/solvent motions, and (3) the temperature range below the glass transition region, for which persistent reaction corresponds to non-native, alternative reaction pathways, in the vicinity of the native configurational envelope. Representative outcomes of low-temperature kinetics studies are portrayed on Eyring and free energy surface (landscape) plots, and guidelines for interpretations are presented.

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“…Classes A, C, and D include active serine enzymes, while class B consists of metalloenzymes. Among the class A enzymes, TEM-1 (263 residues, 28.9 kDa) is by far the most studied by several techniques such as X-ray crystallography ( − ), molecular dynamics (MD) 1 simulations ( − ), electron−nuclear double-resonance spectroscopy (ENDOR) ( , ), and NMR ( − ). Despite all of these studies, aspects of the mechanism of action of catalytic residues in this protein still remain unclear and controversial.…”

mentioning

confidence: 99%

Backbone Dynamics of TEM-1 Determined by NMR: Evidence for a Highly Ordered Protein

Savard

Gagné

2006

Biochemistry

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Backbone dynamics of TEM-1 beta-lactamase (263 amino acids, 28.9 kDa) were studied by 15N nuclear magnetic resonance relaxation at 11.7, 14.1, and 18.8 T. The high quality of the spectra allowed us to measure the longitudinal relaxation rate (R1), the transverse relaxation rate (R2), and the {1H}-15N NOE for up to 227 of the 250 potentially observable backbone amide groups. The model-free formalism was used to determine internal motional parameters using an axially anisotropic model. TEM-1 exhibits a small prolate axial anisotropy (D(parallel)/D(perpendicular) = 1.23 +/- 0.01) and a global correlation time (tau(m)) of 12.41 +/- 0.01 ns. The unusually high average generalized order parameter (S2) of 0.90 +/- 0.02 indicates that TEM-1 is one of the most ordered proteins studied by liquid-state NMR to date. Although the omega-loop has a high degree of order in the picosecond-to-nanosecond time scale (mean S2 value of 0.90 +/- 0.02), we observed the presence of microsecond-to-millisecond time scale motions for this loop, as for the vicinity of the active site. These motions could be relevant for the catalytic function of TEM-1. Amide exchange experiments were also performed, and several amide groups were not exchanged after 12 days, an indication that global motions in TEM-1 are also very limited. Although detailed dynamics characterization by NMR cannot be readily applied to TEM-1 in the presence of relevant substrates, the unusual picosecond-to-nanosecond dynamics behavior of TEM-1 presented here will be essential to the validation and improvement of future molecular dynamics simulations of TEM-1 in the presence of functionally relevant substrates.

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Chromophoric spin-labeled β-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C β-lactamases

Cited by 7 publications

References 38 publications

Kinetic and Thermodynamic Characterization of Co^II−Substrate Radical Pair Formation in Coenzyme B₁₂-Dependent Ethanolamine Ammonia-Lyase in a Cryosolvent System by Using Time-Resolved, Full-Spectrum Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Kinetic and Thermodynamic Characterization of Co^II−Substrate Radical Pair Formation in Coenzyme B₁₂-Dependent Ethanolamine Ammonia-Lyase in a Cryosolvent System by Using Time-Resolved, Full-Spectrum Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems

Backbone Dynamics of TEM-1 Determined by NMR: Evidence for a Highly Ordered Protein

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Chromophoric spin-labeled β-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C β-lactamases

Cited by 7 publications

References 38 publications

Kinetic and Thermodynamic Characterization of CoII−Substrate Radical Pair Formation in Coenzyme B12-Dependent Ethanolamine Ammonia-Lyase in a Cryosolvent System by Using Time-Resolved, Full-Spectrum Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Kinetic and Thermodynamic Characterization of CoII−Substrate Radical Pair Formation in Coenzyme B12-Dependent Ethanolamine Ammonia-Lyase in a Cryosolvent System by Using Time-Resolved, Full-Spectrum Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems

Backbone Dynamics of TEM-1 Determined by NMR: Evidence for a Highly Ordered Protein

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Kinetic and Thermodynamic Characterization of Co^II−Substrate Radical Pair Formation in Coenzyme B₁₂-Dependent Ethanolamine Ammonia-Lyase in a Cryosolvent System by Using Time-Resolved, Full-Spectrum Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Kinetic and Thermodynamic Characterization of Co^II−Substrate Radical Pair Formation in Coenzyme B₁₂-Dependent Ethanolamine Ammonia-Lyase in a Cryosolvent System by Using Time-Resolved, Full-Spectrum Continuous-Wave Electron Paramagnetic Resonance Spectroscopy