Elucidating the Unusual Reaction Kinetics of D-Glucuronyl C5-Epimerase.
Elucidating the Unusual Reaction Kinetics of D-Glucuronyl C5-Epimerase.
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DOI：
10.1093/glycob/cwaa035
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发表时间：
2020-04
期刊：
影响因子：
4.3
通讯作者：
Deepika Vaidyanathan;E. Paskaleva;Troy Vargason;Xia Ke;S. McCallum;R. Linhardt;J. Dordick
中科院分区：
文献类型：
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作者：
Deepika Vaidyanathan;E. Paskaleva;Troy Vargason;Xia Ke;S. McCallum;R. Linhardt;J. Dordick
通过多酶过程的化学酶法合成肝素是为这种动物来源的抗凝血药提供安全有效的替代品所面临的关键挑战。D-葡萄糖醛酸基C5-表异构酶(C5-epi)是一种作用于肝素前体N-磺基肝素的酶，催化D-葡萄糖醛酸(GLCA)可逆地异构化为L-艾杜糖醛酸(IDOA)。由于缺乏可靠的C5-epi检测方法，限制了对酶反应和动力学机制的阐明。介绍了依赖1D1H核磁共振研究C5-epi活性的实时和离线分析方法。首次利用与肝素的化学酶合成和生物合成直接相关的多糖底物，测定了C5-epi正向反应和伪反向反应的表观稳态动力学参数。正向反应表现为异常的S型动力学行为，而伪逆反应表现为非饱和动力学行为。使用一系列缓冲添加剂探索了正向反应的非典型S型行为。令人惊讶的是，每添加25 mM的CaCl2和MgCl2导致了一个正向反应，表现出更传统的米氏动力学。在没有3‘-磷酸腺苷5’-磷酸硫酸盐(PAPS)的情况下，加入参与肝素合成的下一个酶2-O-磺基转移酶，也导致C5-epi在前向反应中表现出更传统的Michaelis-Menten动力学行为，伴随着表观Vmax的显著增加。这项研究为理解C5-epi的反应动力学提供了关键信息，这可能导致改进的化学酶法合成生物工程肝素的方法。
The chemoenzymatic synthesis of heparin, through a multi-enzyme process, represents a critical challenge in providing a safe and effective substitute for this animal sourced anticoagulant drug. D-Glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5-epi has limited elucidation of the enzymatic reaction and kinetic mechanisms. Real time and offline assays are described that rely on 1D 1H NMR to study the activity of C5-epi. Apparent steady-state kinetic parameters for both the forward and the pseudo-reverse reactions of C5-epi are determined for the first time using polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin. The forward reaction shows unusual sigmoidal kinetic behavior and the pseudo-reverse reaction displays non-saturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of buffer additives. Surprisingly, the addition of 25 mM each of CaCl2 and MgCl2 resulted in a forward reaction exhibiting more conventional Michaelis-Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin synthesis, in the absence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), also resulted in C5-epi exhibiting a more conventional Michaelis-Menten kinetic behavior in the forward reaction accompanied by a significant increase in apparent Vmax. This study provides critical information for understanding the reaction kinetics of C5-epi, which may result in improved methods for the chemoenzymatic synthesis of bioengineered heparin.