Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.
Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.
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Xa 因子催化反应的氘溶剂同位素效应和质子库存研究。
DOI：
10.1021/bi061218m
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Kovach,IldikoM
中科院分区：
文献类型：
--
作者：
Zhang,Daoning;Kovach,IldikoM
当底物浓度为0.2 KM时，在Va因子和5×10-5M磷脂微囊存在和不存在的情况下，Xa因子(FxA)催化的凝血酶原激活的动力学溶剂同位素效应(KSIE)略相反，为0.82KSIEs，−为0.93.这与酶−凝血酶原装配的速率决定关联一致，而不是速率限制的化学转化。已知的是，FVA会产生重大的构象变化，暴露凝血酶原中的第一个剪刀键，这可能是触发重大溶剂重排的事件。在凝血酶原浓度为5 kM时，随着FXA和底物浓度的增加，当FXA与FVA的比例为1：1时，KSIE为1.6±0.3，而当FXA/FVA为1：4时，KSIE逐渐减小，分别为0.30±0.05和0.19±0.04。速率决定步骤随条件的变化而变化，但化学步骤在任何情况下都不受限制。这证实了当FXA与凝血酶原复合体充分饱和时，甲硫唑凝血酶途径的优势。相反，FXA催化的N-α-Z-d-Arg-Gly-Arg-PNA·2HCl(S-2765)和H-d-Ile-L-Pro-L-Arg-PNA·HCl(S-2288)最符合在Ser195OγH和His57Nε2与His57Nδ1和AsP102COOβ-之间的过渡态形成的双质子桥，在pH 8.40和25.0±0.1℃的酶饱和条件下，S-2765和S-2288的溶剂重排反应的过渡态分馏因子分别为ϕ1=ϕ2=0.57±0.07和ϕS=0.78±0.16，ϕ1=ϕ2=0.674±0.001。这些反应的速率控制步骤(S)很可能是C−N键的断裂和离开基团的离开。
Kinetic solvent isotope effects (KSIEs) for the factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of factor Va (FVa) and 5.0 × 10-5M phospholipid vesicles are slightly inverse, 0.82−0.93, when substrate concentrations are at 0.2Km. This is consistent with the rate-determining association of the enzyme−prothrombin assembly, rather than the rate-limiting chemical transformation. FVa is known to effect a major conformational change to expose the first scissile bond in prothrombin, which is the likely event triggering a major solvent rearrangement. At prothrombin concentrations > 5Km, the KSIE is 1.6 ± 0.3, when FXa is in a 1:1 ratio with FVa but becomes increasingly inverse, 0.30 ± 0.05 and 0.19 ± 0.04, when FXa/FVa is 1:4, with an increasing FXa and substrate concentration. The rate-determining step changes with the conditions, but the chemical step is not limiting under any circumstance. This corroborates the proposed predominance of the meizothrombin pathway when FXa is well-saturated with the prothrombin complex. In contrast, the FXa-catalyzed hydrolysis of N-α-Z-d-Arg-Gly-Arg-pNA·2HCl (S-2765) and H-d-Ile-l-Pro-l-Arg-pNA·HCl (S-2288) is most consistent with two-proton bridges forming at the transition state between Ser195OγH and His57Nε2 and His57Nδ1 and Asp102COOβ-at the active site, with transition-state fractionation factors of ϕ1= ϕ2= 0.57 ± 0.07 and ϕS= 0.78 ± 0.16 for solvent rearrangement for S-2765 and ϕ1= ϕ2= 0.674 ± 0.001 for S-2288 under enzyme saturation with the substrate at pH 8.40 and 25.0 ± 0.1 °C. The rate-determining step(s) in these reactions is most likely the cleavage of the C−N bond and departure of the leaving group.