Auger M, McDermott A E, Robinson V, Castelhano A L, Billedeau R J, Pliura D H, Krantz A, Griffin R G
Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1993 Apr 20;32(15):3930-4. doi: 10.1021/bi00066a012.
We have used solid-state 13C NMR to study the structure of the adduct resulting from the inactivation of the enzyme transglutaminase by 3-halo-4,5-dihydroisoxazoles. These inhibitors were conceived on the assumption that they would inhibit transglutaminase by attack of an enzyme active site cysteine thiol on the imine carbon of the dihydroisoxazole ring. The tetrahedral intermediate formed could then break down with the loss of the halide group and the subsequent formation of a stable imino thioether adduct. We have compared the 13C CPMAS spectra of the chloro-, bromo-, and (ethylthio)dihydroisozazole inhibitors, and the results indicate that the chemical shift of the C-3 carbon is sensitive to the nature of the heteroatom. Subtraction of the natural-abundance 13C solid-state NMR spectrum of the enzyme from that of the enzyme inactivated by C-3-labeled chlorodihydroisoxazole reveals a broad peak at 156 ppm. The chemical shift of this peak is very close to that observed for a model 3-ethylthio compound and suggests the formation of a stable imino thioether enzyme adduct. Similar results were obtained for lyophilized enzyme adducts and for frozen solutions of the enzyme adduct in the absence and presence of Ca2+. We have also compared these results with those obtained by solution NMR on an aqueous solution of the enzyme-inhibitor complex. The 13C-labeled C-3 resonance was not observed in this case.
我们已使用固态¹³C核磁共振技术来研究由3-卤代-4,5-二氢异恶唑使转谷氨酰胺酶失活所产生的加合物的结构。这些抑制剂是基于这样的假设设计的，即它们会通过酶活性位点的半胱氨酸硫醇对二氢异恶唑环的亚胺碳的攻击来抑制转谷氨酰胺酶。然后形成的四面体中间体可以随着卤化物基团的离去而分解，并随后形成稳定的亚氨基硫醚加合物。我们比较了氯代、溴代和（乙硫基）二氢异恶唑抑制剂的¹³C交叉极化魔角旋转（CPMAS）谱，结果表明C-3碳的化学位移对杂原子的性质敏感。从用C-3标记的氯代二氢异恶唑失活的酶的¹³C固态核磁共振谱中减去酶的天然丰度¹³C固态核磁共振谱，在156 ppm处显示出一个宽峰。该峰的化学位移与模型3-乙硫基化合物观察到的非常接近，表明形成了稳定的亚氨基硫醚酶加合物。对于冻干的酶加合物以及在有无Ca²⁺存在下的酶加合物的冷冻溶液，也获得了类似的结果。我们还将这些结果与通过溶液核磁共振在酶 - 抑制剂复合物的水溶液上获得的结果进行了比较。在这种情况下未观察到¹³C标记的C-3共振。
