Conformational Changes in the Metallo-β-lactamase ImiS During the Catalytic Reaction: An EPR Spectrokinetic Study of Co(II)-Spin Label Interactions

Sharma, Narayan P.; Hu, Zhenxin; Crowder, Michael W.; Bennett, Brian

doi:10.1021/ja0774562

Cited by 17 publications

(31 citation statements)

References 36 publications

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“…A model 715 Update Instruments ram controller was used to drive a PMI-Kollmorgen stepping motor (model 00D12F-02001-1) connected to a ram that in turn drove two Update Instrument syringes of the same inner diameter. The syringes, mixer, and tubing were all contained in a water bath that was maintained at 2 °C [39, 43, 44]. 10 ms intermediate samples were collected in isopentane at −100 °C contained in a glass funnel attached to 4 mm O.D.…”

Section: Methodsmentioning

confidence: 99%

“…The B2 enzymes have an α-helix in the same position as the loop in the B1 and B3 enzymes that appears to have the same function [38, 39]. The helix in the resting state of the B2 MβL CphA has been structurally characterized by X-ray diffraction, but mechanistic data are lacking, whereas an earlier EPR spectrokinetic study of the related enzyme ImiS identified rotation of the helix about its axis during the catalytic cycle [39].…”

Section: Introductionmentioning

confidence: 99%

“…The helix in the resting state of the B2 MβL CphA has been structurally characterized by X-ray diffraction, but mechanistic data are lacking, whereas an earlier EPR spectrokinetic study of the related enzyme ImiS identified rotation of the helix about its axis during the catalytic cycle [39]. The available data suggest that the loop in B1 and B3 MβLs and the position-conserved α-helix in B2 MβLs play a role in substrate binding and catalysis.…”

Section: Introductionmentioning

confidence: 99%

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Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy

et al. 2015

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Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-β-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-β-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20–35 Å away from residue 49, during catalysis. At 10 milliseconds after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy

et al. 2015

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“…However, several methionine residues were identified, including one that resides in the middle of an a-helix that was later shown to be mobile during substrate turnover (89), directly across from a histidine on the surface of the protein. Mutation of this methionine to an isoleucine fully abolished metal ion inhibition in imiS.…”

Section: Mblsmentioning

confidence: 99%

X-Ray Absorption Spectroscopy of Dinuclear Metallohydrolases

Tierney

Schenk

2014

Biophysical Journal

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In this mini-review, we briefly discuss the physical origin of x-ray absorption spectroscopy (XAS) before illustrating its application using dinuclear metallohydrolases as exemplary systems. The systems we have selected for illustrative purposes present a challenging problem for XAS, one that is ideal to demonstrate the potential of this methodology for structure/function studies of metalloenzymes in general. When the metal ion is redox active, XAS provides a sensitive measure of oxidation-state-dependent differences. When the metal ion is zinc, XAS is the only spectroscopic method that will provide easily accessible structural information in solution. In the case of heterodimetallic sites, XAS has the unique ability to interrogate each metal site independently in the same sample. One of the strongest advantages of XAS is its ability to examine metal ion site structures with crystallographic precision, without the need for a crystal. This is key for studying flexible metal ion sites, such as those described in the selected examples, because it allows one to monitor structural changes that occur during substrate turnover.

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“…Stopped-flow fluorescence experiments on L1 (a B3 enzyme) show a catalytically relevant rate of loop movement, further suggesting an important role for the loop in catalysis [23], and EPR studies showed movement of the position-conserved α-helix above the active site of ImiS, which belongs to the B2 class. [24]…”

Section: Introductionmentioning

confidence: 99%

Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies

Aitha¹,

Richmond²,

Hu³

et al. 2014

Journal of Inorganic Biochemistry

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The metallo-β-lactamases (MβLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in β-lactam-containing antibiotics, and render the antibiotics inactive. All known MβLs contain a mobile element near their active sites, and these mobile elements have been implicated in the catalytic mechanisms of these enzymes. However little is known about the dynamics of these elements. In this study, we prepared a site-specific, double spin-labeled analog of homotetrameric MβL L1 with spin labels at position 163 and 286 analyzed the sample with DEER (double electron electron resonance) spectroscopy. Four unique distances were observed in the DEER distance distribution, and these distances were assigned to the desired intramolecular dipolar coupling (between spin labels at positions 163 and 286 in one subunit) and to intermolecular dipolar couplings. To rid the spin-labeled analog of L1 of the intermolecular couplings, spin-labeled L1 was “diluted” by unfolding/refolding the spin-labeled enzyme in the presence of excess wild-type L1. DEER spectra of the resulting, spin-diluted enzyme revealed a single distance corresponding to the desire intramolecular dipolar coupling.

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Conformational Changes in the Metallo-β-lactamase ImiS During the Catalytic Reaction: An EPR Spectrokinetic Study of Co(II)-Spin Label Interactions

Cited by 17 publications

References 36 publications

Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy

Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy

X-Ray Absorption Spectroscopy of Dinuclear Metallohydrolases

Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies

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