Lewis K B, Teller D C, Fry J, Lasser G W, Bishop P D
ZymoGenetics, Inc., Seattle, Washington 98102, USA.
Biochemistry. 1997 Feb 4;36(5):995-1002. doi: 10.1021/bi961636z.
Factor XIII is the terminal enzyme of the coagulation cascade which serves to rapidly crosslink the adjacent gamma-chain C-termini of fibrin clots. In vivo, this process is initiated by the proteolytic action of thrombin which simultaneously converts both soluble fibrinogen to fibrin and activates zymogen FXIII; fibrin then spontaneously polymerizes to form a gel which activated FXIII stabilizes through crosslinking. Due to the kinetic complexity and the difficulty of investigating gel phase reactions, methods employing pre-activation of recombinant human Factor XIII (rFXIII[A'2]) were developed to effectively decouple these reactions. By utilizing these methods, the kinetic parameters of gamma-chain crosslinking in fibrin gels could be determined by both initial rate and integrated rate techniques under physiologically relevant conditions. The crosslinking of the gamma-chain of fibrin gels could be described by apparent Michaelis kinetics with K(m)(app) = 6.2 microM, kcat = 1872 min-1, and Ksp = 302 min-1 microM-1 for a fibrin gamma-chain monomer of M(r) = 170000 Da. In contrast, both the crosslinking rates of alpha-chains within fibrin gels (Ksp = 0.38 min-1 microM-1: Bishop et al. (1993)) and the crosslinking of a soluble synthetic peptide containing the unique gamma-chain fibrin crosslinking site (Ksp = 0.030 min-1 microM-1) could not be shown to saturate and gave apparent first-order rates with respect to rFXIII[A'2]. These observations coupled with the large differences in the turnover rates (approximately 10(4)) suggest two likely mechanisms for FXIII[A'2]-substrate interactions: (1) random (or independent) binding of non- or weakly interacting substrate pairs imposes a high entropic barrier (i. e., delta Gbinding) to the formation of a productive catalytic complex, e.g., for soluble gamma-chain peptides and the flexible alpha-chains within fibrin, and (2) binding to an oriented substrate pair effectively lowers the entropic barrier to formation of a Michaelis complex and thus greatly enhances the rate of catalysis, e.g., for gamma-chain pairs within the fibrin fibrils.
因子 XIII 是凝血级联反应的终末酶，其作用是迅速交联纤维蛋白凝块相邻的γ链 C 末端。在体内，这个过程由凝血酶的蛋白水解作用启动，凝血酶同时将可溶性纤维蛋白原转化为纤维蛋白并激活酶原 FXIII；然后纤维蛋白自发聚合形成凝胶，激活的 FXIII 通过交联使其稳定。由于动力学的复杂性以及研究凝胶相反应的困难，人们开发了采用重组人因子 XIII（rFXIII[A'2]）预激活的方法来有效分离这些反应。通过使用这些方法，在生理相关条件下，可通过初始速率和积分速率技术确定纤维蛋白凝胶中γ链交联的动力学参数。对于分子量为 170000 Da 的纤维蛋白γ链单体，纤维蛋白凝胶γ链的交联可用表观米氏动力学描述，K(m)(app) = 6.2 μM，kcat = 1872 min-1，Ksp = 302 min-1 μM-1。相比之下，纤维蛋白凝胶中α链的交联速率（Ksp = 0.38 min-1 μM-1：Bishop 等人，(1993)）以及含有独特γ链纤维蛋白交联位点的可溶性合成肽的交联（Ksp = 0.030 min-1 μM-1）均未显示出饱和，并且相对于 rFXIII[A'2]呈现表观一级速率。这些观察结果以及周转率的巨大差异（约 10(4)）提示了 FXIII[A'2]与底物相互作用的两种可能机制：(1) 非相互作用或弱相互作用底物对的随机（或独立）结合对形成有催化活性的复合物施加了高熵垒（即，ΔGbinding），例如对于可溶性γ链肽和纤维蛋白内的柔性α链，以及 (2) 与定向底物对的结合有效地降低了形成米氏复合物的熵垒，从而极大地提高了催化速率，例如对于纤维蛋白原纤维内的γ链对。
