Biomedical EPR Part-B Methodology Instrumentation and Dynamics - Sandra R. Eaton

130 DEVKUMAR MUSTAFI AND MARVIN W. MAKINEN

location of the sequestered water, we searched for possible hydrogenbonding contacts that could stabilize a sequestered water molecule within van der Waals hard-sphere constraints satisfying the electron-proton distance of 6.65 ± 0.10 Å and lying close to the molecular plane of the spin-label, as required by the dependence of the resonance features on Accordingly, the H’ resonance was best ascribed to a water molecule hydrogen-bonded to the atoms of the Glu166 side chain, as illustrated in Fig. 22. The ENDOR-defined water molecule sits just on the extended van der Waals surface (Lee and Richards, 1971) of the substrate. The water oxygen lies at an approximate 2.9 Å distance from the carbonyl carbon of the scissile ester bond between the substrate and of Ser70 and forms an angle of ~105° with the C = O bond, precisely that expected for O · · · C = O nucleophilic attack (Burgi et al., 1973, 1974).

Deacylation, as the rate-limiting step for TEM-1 catalyzed hydrolysis of penicillin substrates, is controlled by an ionizing group with precisely that expected for a glutamyl side chain. The results in Figs. 21 and 22 provide direct structural confirmation that the catalytic role of Glu166 is that of a general-base, activating the water molecule for breakdown of the acylenzyme. Moreover, molecular graphics inspection of the active site shows that the group of Lys73, proposed as a general base catalyst for acylation through the action of a hydrogen bonded water molecule (Strynadka et al., 1992), cannot form a hydrogen-bond to the ENDOR defined water molecule in Fig. 22 (the distance of closest approach being ~4 Å). This observation, therefore, rules against the proposed role of Lys73.

On the basis of electrostatic calculations, it has been shown that Glu166 can serve as the general base catalyst in both acylation and deacylation steps of the reaction, catalyzing extraction of the hydroxyl proton of Ser70 to form the acylenzyme and activating a water molecule as the nucleophile for its breakdown (Atanasov et al., 2000). Thus, ENDOR results provide direct confirmation of the role of Glu166 in catalysis and serve to define the structural basis of action. Only in a catalytically competent form of the TEM-1 enzyme has it been possible to identify the hydrolytic water molecule. The results again emphasize the need to structurally characterize true intermediates of enzyme-catalyzed reactions instead of enzymeinhibitor complexes or enzymes rendered catalytically incompetent through mutagenesis.

Figure 22. Stereo view of active site of TEM-1illustrating the ENDOR-defined location of the H’ proton assigned to a sequestered water molecule in the active site. The ENDOR-determined distance from the unpaired electron to one of the water protons and structural relationships to the carboxylate oxygens of Glu166 and to the carbonyl carbon of the ester bond of SLPEN formed with the side chain of Ser70 are indicated. The dotted surface represents the extended van der Waals surface of the spin-labeled acyl group showing that the water molecule is accommodated sterically by the substrate. The star just above the ENDOR-defined water molecule indicates the position of an X-ray-defined water molecule in the free enzyme (Jelsch et al., 1993). Reprinted from Mustafi et at. (2001) with permission.

4.FUTURE PERSPECTIVES AND CONCLUDING REMARKS

In this review we have emphasized characterization of metal-bound and hydrogen-bonded solvent in small molecule and macromolecule environments to highlight the precision and level of structural detail that can be achieved through application of angle-selected ENDOR. The studies reviewed also show that comparable precision can be achieved in defining structure and conformation of active site residues and of the substrate in cryokinetically isolated reaction intermediates of enzymes. In this respect, ENDOR spectroscopy applied in conjunction with cryosolvent methods offers many advantages that cannot be achieved through application of other spectroscopic methods capable of three-dimensional structure determination, for instance, multi-dimensional NMR. While the latter is well suited for defining the relative spatial distribution of atoms in a protein in solution (at present 30 kDa) through nuclear Overhauser measurements, given the covalent bonding structure of the constituent amino acid residues, the uncertainties are significantly larger than in ENDOR, up to 50%, for internuclear separations 5.0 Å (Zhao and Jardetzky, 1994; Gradwell and

Feeney, 1996; Zabell and Post, 2002). Furthermore, the time required for NMR data collection of macromolecules in solution and the viscosity of cryosolvent mixtures at low temperatures are incompatible with structural analysis of true intermediates of enzyme-catalyzed reactions or of other chemically labile systems.

Application of cw ENDOR is associated, nonetheless, with inherent limitations despite the high precision afforded for structure analysis. Threedimensional structure determination by cw ENDOR is most straightforwardly carried out with I = 1/2 nuclei. Assignments of hydrogen resonances are generally dependent on carrying out synthetic chemical procedures for site-specific incorporation of deuterium to be used in parallel experiments. Such added chemical complexity is often time-consuming and arduous. In addition, signal-to-noise in data collection becomes an important consideration since the intensity of resonance features is dependent not only on the electron-nucleus distance, anisotropy of relaxation processes, and the number of nuclei contributing to the resonance, but also on the nuclear moment. Because of this latter factor, use of an important nuclide in multi-dimensional NMR experiments, has been limited in cw ENDOR, and most cw ENDOR studies have been restricted to and for structural analysis of the type described here. While improved resolution of overlapping resonance features may be achieved by cw ENDOR instrumentation applied at higher microwave frequencies through greater separation of g-values, structure analysis is not likely to be significantly improved unless the number of magnetic nuclei in the molecule of interest used for structure analysis can be increased.

Because the number of electron-nucleus distances determined in cw ENDOR experiments is relatively small, structure analysis at present rests heavily on bond distance and valence angle information determined independently for large fragments of the molecular complex when ENDORdetermined electron-nucleus distances are applied as constraints in torsion angle search calculations. These fragments provide the molecular scaffolding to which the “ENDOR-active” nuclei are covalently attached. Improvement in structure analysis beyond that achieved hitherto with cw ENDOR, therefore, requires a means to increase significantly the number of independently determined electron-nucleus distances. Of most importance, therefore, are pulsed EPR and ENDOR methods that can provide a means to increase the number of electron-nucleus distances through detection of

and since these nuclides are not easily employed in cw ENDOR. Two recent reviews describe pulsed EPR and ENDOR spectroscopy and their applications to biological systems (Cammack et al., 1999; Prisner et al., 2001). Also, a series of investigations have been reported in which pulsed EPR and ENDOR methods have been applied to

characterize structures of enzyme active sites and to determine proteinligand interactions (Gurbiel, et al, 1996; Gromov et al., 1999; Walsby et al., 2001). Pulsed methods have also been used to characterize structured solvent in macromolecular systems (Goldfarb, et al., 1996; DeRose et al., 1996).

With respect to the objective of increasing the number of dipolar electron-nucleus distances through a combination of cw and pulsed EPR and ENDOR methods, the goal in structural analysis should be to decrease as much as possible the dependence of the analysis on stereochemical data that define the molecular scaffold to which the “ENDOR-active” nuclei are covalently attached. For instance, while the conformation of SLCEP in Fig. 14 was assigned successfully through use of ENDOR-determined distance constraints, the analysis relies heavily on input from X-ray diffraction studies providing bond distances and valence angles of atoms that are not ENDOR-active. In this respect, it should be noted that direct detection of NMR in the vicinity of paramagnetic centers is favored through paramagnetic relaxation processes in contrast to (Banci et al., 1991). On this basis it may be possible to take advantage of the differential relaxation characteristics of and with stochastic ENDOR (Brueggeman and Niklas, 1994). This is essentially a pulsed ENDOR technique. While approaches to extract geometrical information based on this differential property have yet to be developed, it has been shown that NMR detection of near paramagnetic centers in proteins can be used to identify residue connectivities (Machonkin et al., 2002).

The three-dimensional structure and conformation of a (diamagnetic) chemotactic tripeptide uniformly enriched with and has been determined on the basis of simulated annealing calculations restrained by inter-nuclear distance and torsion angle measurements obtained through solid-state, magic-angle spinning NMR experiments (Rienstra et al., 2002). Extension of a similar approach should be feasible for paramagnetic solidstate systems through a combination of cw and pulsed EPR and ENDOR methods. This combined approach need not be restricted only to macromolecules with naturally occurring paramagnetic sites. Applications of ENDOR structural probes such as nitroxyl spin-labels and the cation would be of clear advantage in view of the success with which they have been employed in both small molecule and macromolecular systems hitherto. On this basis, combined application of cw and pulsed ENDOR methods is likely to yield a significantly enhanced basis for structure assignments.

5.ACKNOWLEDGMENTS

This work has been supported by grants of the National Science Foundation (MCB-0092524) and of the National Institutes of Health (DK57599).

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