A novel iterative procedure is described that allows both the orientation and dynamics of internuclear bond vectors to be determined from direct interpretation of NMR dipolar couplings, measured under at least three orthogonal alignment conditions. If five orthogonal alignments are available, the approach also yields information on the degree of motional anisotropy and the direction in which the largest amplitude internal motion of each bond vector takes place. The method is demonstrated for the backbone (15)N-(1)H, (13)C(alpha)-(1)H(alpha), and (13)C(alpha)-13C' interactions in the previously well-studied protein domain GB3, dissolved in a liquid crystalline suspension of filamentous phage Pf1. Alignment variation is achieved by using conservative mutations of charged surface residues. Results indicate remarkably uniform backbone dynamics, with amplitudes that agree well with those of previous (15)N relaxation studies for most residues involved in elements of secondary structure, but larger amplitude dynamics than those found by (15)N relaxation for residues in loop and turn regions. In agreement with a previous analysis of dipolar couplings, the N-H bonds in the second beta-strand, which is involved in antibody recognition, show elevated dynamics with largest amplitudes orthogonal to the chain direction.
The N-H bond length in backbone peptide groups of the protein GB3 has been studied by liquid crystal NMR, using five structurally conserved mutants of this protein. In the absence of additional information, the impact of dynamic fluctuations of the N-H vector orientation on the 15 N-1 H dipolar interaction cannot be separated from a change in N-H bond length. However, a change in N-H bond E-mail: E-mail: bax@nih.gov. Supporting Information Available: Theoretical analysis of positional distribution of H N under zero-point motion; calculated impact of H-bonding on r NH eq ; plot of experimental 3 J HNHα vs DIDC-derived dihedral angle; tables with RDC values. (Fig. 1A): The equilibrium bond length, r NH eq , corresponds to the internuclear distance of lowest energy. The average
Site-specific 15 N chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide 15 N nuclei in the B3 domain of protein G (GB3) from residual chemical shift anisotropy (RCSA) measured in six different mutants that retain the native structure but align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension. This information is complemented by measurement of cross-correlated relaxation rates between the 15 N CSA tensor and either the 15 N-1 H or 15 N-13 C′ dipolar interaction. In agreement with recent solid state NMR measurements, the 15 N CSA tensors exhibit only a moderate degree of variation from averaged values, but have larger magnitudes in α-helical (−173 ± 7 ppm) than in β-sheet (−162 ± 6 ppm) residues, a finding also confirmed by quantum computations. The orientations of the least shielded tensor component cluster tightly around an in-peptide-plane vector that makes an angle of 19.6±2.5° with the N-H bond, with the asymmetry of the 15 N CSA tensor being slightly smaller in α-helix (η=0.23±0.17) than in β-sheet (η=0.31±0.11). The residue-specific 15 N CSA values are validated by improved agreement between computed and experimental 15 N R 1ρ relaxation rates measured for 15 N-{ 2 H} sites in GB3, which are dominated by the CSA mechanism. Use of residuespecific 15 N CSA values also results in more uniform generalized order parameters, S 2 , and predicts considerable residue-by-residue variations in the magnetic field strengths where TROSY line narrowing is most effective.
The complete path for the deamination reaction catalyzed by yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, has been investigated using the ONIOM method. Cytosine deamination proceeds via a sequential mechanism involving the protonation of N(3), the nucleophilic attack of C(4) by the zinc-coordinated hydroxide, and the cleavage of the C(4)-N(4) bond. The last step is the rate determining step for the generation of the zinc bound uracil. Uracil is liberated from the Zn atom by an oxygen exchange mechanism that involves the formation of a gem-diol intermediate from the Zn bound uracil and a water molecule, the C(4)-O(Zn) cleavage, and the regeneration of the Zn-coordinated water. The rate determining step in the oxygen exchange is the formation of the gem-diol intermediate, which is also the rate determining step for the overall yCD-catalyzed deamination reaction.
We report a unique strategy for the development of a H O -dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non-native substrates with the assistance of dual-functional small molecules (DFSMs), such as N-(ω-imidazolyl fatty acyl)-l-amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid-base catalyst in the activation of H O . This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450-H O system previously reported. This work provides the first example of the activation of the normally H O -inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.
The presence of dipole-dipole cross-correlated relaxation as well as unresolved E.COSY effects adversely impacts the accuracy of 1 J NH splittings measured from gradient-enhanced IPAP-HSQC spectra. For isotropic samples, the size of the systematic errors caused by these effects depends on the values of 2 J NHα , 3 J NHβ and 3 J HNHα . Insertion of band-selective 1 H decoupling pulses in the IPAP-HSQC experiment eliminates these systematic errors and for the protein GB3 yields 1 J NH splittings that agree to within a root-mean-square difference of 0.04 Hz with values measured for perdeuterated GB3. Accuracy of the method is also highlighted by a good fit to the GB3 structure of the 1 H-15 N RDCs extracted from the minute differences in 1 J NH splitting measured at 500 and 750 MHz 1 H frequencies, resulting from magnetic susceptibility anisotropy. A nearly complete set of 2 J NHα couplings was measured in GB3 in order to evaluate whether the impact of cross-correlated relaxation is dominated by the 15 N-1 H α or 15 N-1 H β dipolar interaction. As expected, we find that 2 J NHα ≤2 Hz, with values in the α-helix (0.86 ± 0.52 Hz) slightly larger than in β-sheet (0.66 ± 0.26 Hz). Results indicate that under isotropic conditions, N-H N /N-H β cross-correlated relaxation often dominates. Unresolved E.COSY effects under isotropic conditions involve 3 J HNHα and J NHα , but when weakly aligned any aliphatic proton proximate to both N and H N can contribute.
Yeast cytosine deaminase (yCD), a zinc metalloenzyme, catalyzes the hydrolytic deamination of cytosine to uracil. The enzyme is of great biomedical interest because it also catalyzes the deamination of the prodrug 5-fluorocytosine (5FC) to form the anticancer drug 5-fluorouracil (5FU). yCD/5FC is one of the most widely used enzyme/prodrug combinations for gene-directed enzyme prodrug therapy for the treatment of cancers. A pH indicator assay has been developed for the measurement of the steady-state kinetic parameters for the deamination reaction. Transient kinetic studies have shown that the product release is a rate-limiting step in the activation of the prodrug 5FC by yCD. The rate constant of the chemical step for the forward reaction (250 s(-)(1)) is approximately 8 times that of the product release (31 s(-)(1)) and approximately 15 times k(cat) (17 s(-)(1)). The transient kinetic results are consistent with those of the steady-state kinetic analysis in the sense that the k(cat) and K(m) values calculated from the rate constants determined by the transient kinetic analysis are in close agreement with those measured by the steady-state kinetic analysis. NMR experiments have demonstrated that free 5FU is in slow exchange with its complex with yCD but has a low affinity for yCD. The transient kinetic and NMR results together suggest that the release of 5FU is rate-limiting in the activation of the prodrug 5FC by yCD and may involve multiple steps.
Site-specific 1H chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide moieties in the B3 domain of protein G (GB3). Experimental input data include residual chemical shift anisotropy (RCSA), measured in six mutants that align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension, and cross-correlated relaxation rates between the 1HN CSA tensor and either the 1H−15N, the 1H−13C′, or the 1H−13Cα dipolar interactions. Analyses with the assumption that the 1HN CSA tensor is symmetric with respect to the peptide plane (three-parameter fit) or without this premise (five-parameter fit) yield very similar results, confirming the robustness of the experimental input data, and that, to a good approximation, one of the principal components orients orthogonal to the peptide plane. 1HN CSA tensors are found to deviate strongly from axial symmetry, with the most shielded tensor component roughly parallel to the N−H vector, and the least shielded component orthogonal to the peptide plane. DFT calculations on pairs of N-methyl acetamide and acetamide in H-bonded geometries taken from the GB3 X-ray structure correlate with experimental data and indicate that H-bonding effects dominate variations in the 1HN CSA. Using experimentally derived 1HN CSA tensors, the optimal relaxation interference effect needed for narrowest 1HN TROSY line widths is found at ∼1200 MHz.
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