Stevens W K, Nesheim M E
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Biochemistry. 1993 Mar 23;32(11):2787-94. doi: 10.1021/bi00062a008.
Increases in intrinsic fluorescence (delta I), reflecting changes in tryptophan environments, occur upon bond cleavages necessary for prothrombin (II) activation to thrombin (IIa) by prothrombinase. Cleavage at Arg274-Thr275 (numbering based on bovine prothrombin sequence, with chymotrypsinogen numbering in braces) between the amino-terminal fragment 1.2 and protease (Pre2) domains of prothrombin yields delta I = 5%, and cleavage within the Pre2 domain at Arg323-Ile324 to form IIa yields delta I = 35%, while cleavage at both yields delta I = 25%. Since the change in fluorescence upon activation of prothrombin can be largely attributed to a change within the Pre2 domain, the susceptibilities of each of the 9 Trp residues of IIa and its immediate precursor Pre2 to oxidation by N-bromosuccinimide (NBS) were compared. Pre2 and IIa were titrated with increasing amounts of NBS (0.5-5 equiv of NBS/TRP), aliquots were removed and fully digested with trypsin, and tryptophan-containing peptides were separated and quantitated by RP-HPLC with fluorescence detection. Tryptic digests yielded 9 tryptophan-containing peptides, which were identified by amino acid composition. Tryptophan residues in IIa and Pre2 displayed a 10-fold range of sensitivity to modification. Tryptophans 337 and 360 (W29, W51) were modified less readily in IIa than in Pre2, while residues 373, 542, and 550 (W60D, W207, W215) were modified more readily, and other residues were equally susceptible. Residues 360 and 373 (W29, W60D) flank the active site histidine. From the crystal structure, residues 373 and 550 (W60D, W215) are implicated in substrate binding.(ABSTRACT TRUNCATED AT 250 WORDS)
凝血酶原酶将凝血酶原（II）激活为凝血酶（IIa）所需的键断裂时，会发生固有荧光增加（ΔI），这反映了色氨酸环境的变化。凝血酶原在氨基末端片段1.2和蛋白酶（Pre2）结构域之间的Arg274 - Thr275（编号基于牛凝血酶原序列，括号内为胰凝乳蛋白酶原编号）处裂解产生ΔI = 5%，在Pre2结构域内Arg323 - Ile324处裂解形成IIa产生ΔI = 35%，而在两处均裂解产生ΔI = 25%。由于凝血酶原激活时荧光的变化很大程度上归因于Pre2结构域内的变化，因此比较了IIa及其直接前体Pre2的9个色氨酸残基对N - 溴代琥珀酰亚胺（NBS）氧化的敏感性。用递增剂量的NBS（0.5 - 5当量的NBS/TRP）滴定Pre2和IIa，取出等分试样并用胰蛋白酶完全消化，含色氨酸的肽通过反相高效液相色谱结合荧光检测进行分离和定量。胰蛋白酶消化产生9个含色氨酸的肽，通过氨基酸组成进行鉴定。IIa和Pre2中的色氨酸残基对修饰的敏感性范围为10倍。色氨酸337和360（W29，W51）在IIa中比在Pre2中更不易被修饰，而残基373、542和550（W60D，W207，W215）更易被修饰，其他残基的敏感性相同。残基360和373（W29，W60D）位于活性位点组氨酸两侧。从晶体结构来看，残基373和550（W60D，W215）与底物结合有关。（摘要截断于250字）
