Solid state NMR studies of hydrogen bonding in a citrate synthase inhibitor complex

The ionization state and hydrogen bonding environment of the transition sta te 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 contr ibution of short hydrogen bonds to binding energies and enzyme catalysis: t he X-ray crystal structure of this complex revealed an unusually short hydr ogen bond between the carboxylate group of the inhibitor and an aspartic ac id side chain [Usher et al. (1994) Biochemistry 33, 7753-7759]. To further investigate the nature of this short hydrogen bond, low spinning speed C-13 NMR spectra of the CMX-citrate synthase complex were obtained under a vari ety of sample conditions. Tensor values describing the chemical shift aniso tropy of the carboxyl groups of the inhibitor were obtained by simulating M AS spectra (233 +/- 4, 206 +/- 5, and 105 +/- 2 ppm vs TMS). Comparison of these values with our previously reported database and ab initio calculatio ns of carbon shift tensor values clearly indicates that the carboxyl is dep rotonated. New data from model compounds suggest that hydrogen bonds in a s yn arrangement with respect to the carboxylate group have a pronounced effe ct upon the shift tensors for the carboxylate, while anti hydrogen bonds, r egardless of their length, apparently do not perturb the shift tensors of t he 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 hy drogen bond; the latter would agree very well with previous crystallographi c results. Two-dimensional H-1-C-13 heteronuclear correlation spectra of th e enzyme-inhibitor complex were obtained. Strong cross-peaks were observed from the carboxyl carbon to proton(s) with chemical shift(s) of 22 +/-. 5 p pm. Both the proton chemical shift and the intensity of the cross-peak indi cate 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 Angs trom. This proton resonance is assigned to H-delta 2 of Asp 375, on the bas is 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 carbo xylate.