SOYBEAN TRYPSIN-INHIBITOR (KUNITZ) AND ITS COMPLEX WITH TRYPSIN - CARBON-13 NUCLEAR MAGNETIC-RESONANCE STUDIES OF THE REACTIVE SITE ARGININE
SOYBEAN TRYPSIN-INHIBITOR (KUNITZ) AND ITS COMPLEX WITH TRYPSIN - CARBON-13 NUCLEAR MAGNETIC-RESONANCE STUDIES OF THE REACTIVE SITE ARGININE
复制标题
DOI：
10.1021/bi00566a006
复制
发表时间：
1980-01-01
期刊：
影响因子：
2.9
通讯作者：
MARKLEY, JL
中科院分区：
文献类型：
--
作者：
BAILLARGEON, MW;LASKOWSKI, M;MARKLEY, JL
The reactive site Arg63-Ile64 peptide bond of soybean trypsin inhibitor (STI) is preferentially and reversibly hydrolyzed by trypsin. A modified method was used to replace Arg63 of STI with [13C.zeta.]arginine and [13C.degree.]arginine. Protein purity was established by disc gel electrophoresis. The 13C NMR spectrum of [[13C.zeta.]Arg63]STI revealed a single peak with chemical shift 157.81 ppm [downfield from (CH3)4Si] above the background spectrum of unenriched STI. When sufficient bovine or porcine trypsin was added to complex all but a small fraction of the [[13C.zeta.]Arg63]STI, the peak assigned to Arg63 C.zeta. shifted to 158.04 ppm. Apparently, from the negligible chemical shift change, there is no signficant proton transfer from the guanidinum group upon complex formation. The 13C NMR spectrum of [[13C.degree.]Arg63]STI revealed 1 peak at 173.8 ppm. In labeled modified STI, [[13C.degree.]-Arg63]STI(63,64), the chemical shift of the labeled carbon was pH dependent (179.0 ppm at pH 7.0; 174.7 ppm at pH 2.9). When a half-stoichiometric amount of bovine trypsin was added to labeled STI, 2 13C NMR peaks were observed, at 173.8 and 174.7 ppm. Disc gel electrophoresis identified the 2 species as [[13C.degree.]Arg63]STI and the complex, [[13C.degree.]-Arg63]STI-trypsin. After treatment with sufficient bovine trypsin to yield only a small excess of labeled STI, a single peak remained at 174.7 ppm. The absence of a large upfield shift of the [13C.degree.]Arg63 peak upon complexation establishes that the STI-trypsin complex is not a covalent, fully tetrahedral adduct. A covalent, fully tetrahedral intermediate structure has been proposed for the STI-trypsin complex. Although a covalent, fully tetrahedral structure was proposed initially for the pancreatic trypsin inhibitor-trypsin complex, subsequent X-ray experiments have ruled this out. The present 13C NMR results do not allow us to distinguish between a complex in which C.degree. is completely trigonal and the structure recently proposed for PTI-trypsin based on X-ray studies, where C.degree. is partially tetrahedral due to constraints imposed by the enzyme-inhibitor contact.
