用N-亚硝基-N-苄基乙酰胺及相关化合物标记α-糜蛋白酶的活性位点。

Labeling of the active site of alpha-chymotrypsin with N-nitroso-N-benzylacetamide and related compounds.

作者信息

Li M, Roswell D F, White E H

机构信息

Department of Chemistry, Johns Hopkins University Baltimore, Maryland 21218.

出版信息

Biochem Biophys Res Commun. 1993 Oct 29;196(2):907-13. doi: 10.1006/bbrc.1993.2335.

DOI:10.1006/bbrc.1993.2335

PMID:8240368

Abstract

alpha-Chymotrypsin (CT) is slowly inhibited by N-nitroso-N-benzylacetamide (1) at 25 degrees C. The 13C-NMR spectrum of the hydrolysate of 13C-enriched 1-inhibited CT shows five new signals at 52.33, 44.55, 43.72, 36.79, and 32.90 ppm resulting from alkylation of the side chains and also of amide linkages of the enzyme backbone by benzyl carbocations produced in the active site. The alkylation pattern is different from those observed in the inhibitions of CT with D-N-nitroso-N-[alpha-13C]-benzyl-N'- isobutyrylalaninamide (2a) and D-N-nitroso-N-[alpha-13-C]-benzyl-N'-isobutyrylphenylalaninamide (2b) (White et al. JACS 1990, 112, 1956-1961). The chemical shift data suggest that the 52.33 ppm signal stems from N-benzylglycine, the 36.79 ppm signal from S-benzylcysteine, and the 32.90 ppm signal from 2-benzyltryptophan. A synthesis of the latter compound was developed.

摘要

α-胰凝乳蛋白酶（CT）在25℃下会被N-亚硝基-N-苄基乙酰胺（1）缓慢抑制。富含13C的1-抑制型CT水解产物的13C-NMR谱在52.33、44.55、43.72、36.79和32.90 ppm处显示出五个新信号，这些信号源于活性位点产生的苄基碳正离子对酶主链侧链以及酰胺键的烷基化作用。这种烷基化模式与用D-N-亚硝基-N-[α-13C]-苄基-N'-异丁酰丙氨酰胺（2a）和D-N-亚硝基-N-[α-13-C]-苄基-N'-异丁酰苯丙氨酰胺（2b）抑制CT时观察到的模式不同（怀特等人，《美国化学会志》，1990年，112卷，1956 - 1961页）。化学位移数据表明，52.33 ppm处的信号源于N-苄基甘氨酸，36.79 ppm处的信号源于S-苄基半胱氨酸，32.90 ppm处的信号源于2-苄基色氨酸。已开发出后一种化合物的合成方法。