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  • Title: Solid state NMR studies of hydrogen bonding in a citrate synthase inhibitor complex.
    Author: Gu Z, Drueckhammer DG, Kurz L, Liu K, Martin DP, McDermott A.
    Journal: Biochemistry; 1999 Jun 22; 38(25):8022-31. PubMed ID: 10387046.
    Abstract:
    The ionization state and hydrogen bonding environment of the transition state analogue (TSA) inhibitor, carboxymethyldethia coenzyme A (CMX), bound to citrate synthase have been investigated using solid state NMR. This enzyme-inhibitor complex has been studied in connection with the postulated contribution of short hydrogen bonds to binding energies and enzyme catalysis: the X-ray crystal structure of this complex revealed an unusually short hydrogen bond between the carboxylate group of the inhibitor and an aspartic acid side chain [Usher et al. (1994) Biochemistry 33, 7753-7759]. To further investigate the nature of this short hydrogen bond, low spinning speed 13C NMR spectra of the CMX-citrate synthase complex were obtained under a variety of sample conditions. Tensor values describing the chemical shift anisotropy of the carboxyl groups of the inhibitor were obtained by simulating MAS spectra (233 +/- 4, 206 +/- 5, and 105 +/- 2 ppm vs TMS). Comparison of these values with our previously reported database and ab initio calculations of carbon shift tensor values clearly indicates that the carboxyl is deprotonated. New data from model compounds suggest that hydrogen bonds in a syn arrangement with respect to the carboxylate group have a pronounced effect upon the shift tensors for the carboxylate, while anti hydrogen bonds, regardless of their length, apparently do not perturb the shift tensors of the carboxyl group. Thus the tensor values for the enzyme-inhibitor complex could be consistent with either a very long syn hydrogen bond or an anti hydrogen bond; the latter would agree very well with previous crystallographic results. Two-dimensional 1H-13C heteronuclear correlation spectra of the enzyme-inhibitor complex were obtained. Strong cross-peaks were observed from the carboxyl carbon to proton(s) with chemical shift(s) of 22 +/- 5 ppm. Both the proton chemical shift and the intensity of the cross-peak indicate a very short hydrogen bond to the carboxyl group of the inhibitor, the C.H distance based upon the cross-peak intensity being 2.0 +/- 0.4 A. This proton resonance is assigned to Hdelta2 of Asp 375, on the basis of comparison with crystal structures and the fact that this cross-peak was absent in the heteronuclear correlation spectrum of the inhibitor-D375G mutant enzyme complex. In summary, our NMR studies support the suggestion that a very short hydrogen bond is formed between the TSA and the Asp carboxylate.
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