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首页> 外文期刊>Biochemistry >Solid state NMR studies of hydrogen bonding in a citrate synthase inhibitor complex.
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Solid state NMR studies of hydrogen bonding in a citrate synthase inhibitor complex.

机译：柠檬酸合酶抑制剂复合物中氢键的固态NMR研究。

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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.
机译：已使用固态NMR研究了过渡态类似物（TSA）抑制剂，与柠檬酸合酶结合的羧甲基硫代辅酶A（CMX）的电离态和氢键环境。已经研究了这种酶-抑制剂配合物与短氢键对结合能和酶催化的假定贡献有关：这种配合物的X射线晶体结构表明，抑制剂的羧酸盐基团和天冬氨酸之间存在异常短的氢键。酸性侧链[Usher等。 （1994）生物化学33，7753-7759]。为了进一步研究这种短氢键的性质，在各种样品条件下获得了CMX柠檬酸合酶复合物的低自旋速度13 C NMR光谱。通过模拟MAS谱图获得了描述抑制剂羧基化学位移各向异性的张量值（相对于TMS为233 +/- 4、206 +/- 5和105 +/- 2 ppm）。将这些值与我们先前报告的数据库进行比较，以及碳位移张量值的从头算起，可以清楚地表明羧基已去质子化。来自模型化合物的新数据表明，相对于羧酸根基团呈顺式排列的氢键对羧酸根的位移张量具有显着影响，而无论氢键的长度如何，反氢键显然都不会干扰羧酸根的位移张量。羧基。因此，酶-抑制剂复合物的张量值可能与很长的合成氢键或反氢键一致。后者与先前的晶体学结果非常吻合。获得了酶-抑制剂复合物的二维1H-13C异核相关光谱。观察到从羧基碳到质子的强交叉峰，化学位移为22 +/- 5 ppm。质子化学位移和交叉峰强度都表明与抑制剂羧基的氢键非常短，基于交叉峰强度的CH距离为2.0 +/- 0.4A。根据与晶体结构的比较以及抑制剂-D375G突变酶复合物的异核相关光谱中不存在该交叉峰的事实，确定了Asp 375的Hdelta2。总而言之，我们的NMR研究支持以下建议：在TSA和Asp羧酸盐之间形成非常短的氢键。

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