Rezaie A R, He X
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA.
Biochemistry. 2000 Feb 22;39(7):1817-25. doi: 10.1021/bi992006a.
The nature of residue 225 on a consensus loop in serine proteases determines whether a protease can bind Na(+). Serine proteases with a Pro at this position are unable to bind Na(+), but those with a Tyr or Phe can bind Na(+). Factor Xa (FXa), the serine protease of the prothrombinase complex, contains a Tyr at this position. Na(+) is also known to stimulate the amidolytic activity of FXa toward cleavage of small synthetic substrates, but the role of Na(+) in the prothrombinase complex has not been investigated. In this study, we engineered a Gla-domainless form of FX (GDFX) in which residue Tyr(225) was replaced with a Pro. We found that Na(+) stimulated the cleavage rate of chromogenic substrates by FXa or GDFXa approximately 8-24-fold with apparent dissociation constants [K(d(app))] of 37 and 182 mM in the presence and absence of Ca(2+), respectively. In contrast, Na(+) minimally affected the cleavage rate of these substrates by the mutant, and no K(d(app)) for Na(+) binding to the mutant could be estimated. Unlike the wild-type enzyme, the reactivity of the mutant with antithrombin was independent of Na(+) and impaired approximately 32-fold. Ca(2+) improved the reactivity of the mutant with antithrombin approximately 5-fold. Affinity of the mutant for binding to factor Va was weakened and its ability to activate prothrombin was severely impaired. Further studies with the wild-type prothrombinase complex revealed that FXa binds to factor Va with a similar K(d(app)) of 1. 1-1.8 nM in the presence of Na(+), K(+), Li(+), Ch(+), and Tris(+) and that the catalytic efficiency of prothrombinase is enhanced less than 1.5-fold by the specific effect of Na(+) in the reaction buffer. These results suggest that (1) the loop including residue 225 (225-loop) is a Na(+) binding site in FXa, (2) the Na(+)- and Ca(2+)-binding loops of FXa are allosterically linked, and (3) the Tyr conformer of the 225-loop is critical for factor Xa function; however, both Na(+)-bound and Na(+)-free forms of factor Xa in the prothrombinase complex can efficiently activate prothrombin.
丝氨酸蛋白酶共有序列环上第225位残基的性质决定了该蛋白酶是否能结合Na⁺。此位置为脯氨酸的丝氨酸蛋白酶无法结合Na⁺，但那些此位置为酪氨酸或苯丙氨酸的则能结合Na⁺。凝血酶原酶复合物的丝氨酸蛋白酶因子Xa（FXa）在此位置含有一个酪氨酸。已知Na⁺能刺激FXa对小分子合成底物切割的酰胺水解活性，但Na⁺在凝血酶原酶复合物中的作用尚未得到研究。在本研究中，我们构建了一种无Gla结构域的FX形式（GDFX），其中第225位残基酪氨酸被脯氨酸取代。我们发现，在有Ca²⁺和无Ca²⁺存在时，Na⁺分别使FXa或GDFXa对显色底物的切割速率提高约8 - 24倍，其表观解离常数[K(d(app))]分别为37 mM和182 mM。相比之下，Na⁺对该突变体切割这些底物的速率影响极小，且无法估算Na⁺与该突变体结合的K(d(app))。与野生型酶不同，该突变体与抗凝血酶的反应性与Na⁺无关，且降低了约32倍。Ca²⁺使该突变体与抗凝血酶的反应性提高了约5倍。该突变体与因子Va结合的亲和力减弱，其激活凝血酶原的能力严重受损。对野生型凝血酶原酶复合物的进一步研究表明，在存在Na⁺、K⁺、Li⁺、Ch⁺和Tris⁺时，FXa与因子Va结合的K(d(app))相似，为1.1 - 1.8 nM，并且反应缓冲液中Na⁺的特异性作用使凝血酶原酶的催化效率提高不到1.5倍。这些结果表明：（1）包含第225位残基的环（225环）是FXa中的一个Na⁺结合位点；（2）FXa的Na⁺结合环和Ca²⁺结合环通过别构作用相连；（3）225环的酪氨酸构象对因子Xa的功能至关重要；然而，凝血酶原酶复合物中结合Na⁺和未结合Na⁺的因子Xa形式均能有效激活凝血酶原。
