Structural changes in the protease domain of prothrombin upon activation as assessed by N-bromosuccinimide modification of tryptophan residues in prethrombin-2 and thrombin.
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)
Stevens
W K
WK
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Nesheim
M E
ME
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D005165:Factor V / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism; D014357:Trypsin; D014364:Tryptophan
D004792:Enzyme Precursors; D010446:Peptide Fragments; D006639:Histidine; D014364:Tryptophan; D005165:Factor V; D011516:Prothrombin; D005170:Factor X; D013925:Thromboplastin; C012007:prethrombins; D010450:Endopeptidases; D014357:Trypsin; D013917:Thrombin; D001974:Bromosuccinimide