Mapping of the catalytic groove preferences of factor Xa reveals an inadequate selectivity for its macromolecule substrates

被引:53
作者
Bianchini, EP
Louvain, VB
Marque, PE
Juliano, MA
Juliano, L
Le Bonniec, BF
机构
[1] Univ Paris 05, Fac Pharm, INSERM, U428, F-75270 Paris 06, France
[2] Escola Paulista Med, Dept Biophys, BR-04044020 Sao Paulo, Brazil
关键词
D O I
10.1074/jbc.M201139200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Factor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P-3-P-2- P-1-P-1' residues, but the exact preferences of its catalytic groove remain largely unknown. To investigate the specificity of FXa, we synthesized full sets of fluorescence-quenched substrates carrying all natural amino acids (except Cys) in P-3, P-2, P-1', P-2', and P-3' and determined the k(cat)/K-m values of cleavage. Contrary to expectation, glycine was not the "best" P-2 residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P-2, the ratio of the k(cat)/K-m values for the most favorable side chain over the least was 289 (12 with trypsin), but in P-1', this ratio was only 30 (versus 80 with trypsin). This unexpected selectivity undoubtedly distinguished FXa from thrombin, which exhibited ratios higher than 19,000 in P-2 and P-1'. Thus, with respect to the catalytic groove, FXa resembles a low efficiency trypsin rather than the highly selective thrombin. The rates of cleavage of the peptidyl substrates were virtually identical whether or not FXa was in complex with factor Va, suggesting that the cofactor did not exert a direct allosteric control on the catalytic groove. We conclude that the remarkable efficacy of FXa within prothrombinase originates from exosite interaction(s) with factor Va and/or prothrombin rather than from the selectivity of its catalytic groove.
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页码:20527 / 20534
页数:8
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