Kinetics of iterative carbohydrate transfer to polysaccharide catalyzed by chondroitin polymerase on a highly sensitive flow-type 27 MHz quartz-crystal microbalance.
Kinetics of iterative carbohydrate transfer to polysaccharide catalyzed by chondroitin polymerase on a highly sensitive flow-type 27 MHz quartz-crystal microbalance.
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DOI：
10.1002/chem.201200342
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发表时间：
2012-06
期刊：
影响因子：
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通讯作者：
Toshiaki Mori;T. Kodera;Hiroshi Yoshimine;Y. Kakuta;N. Sugiura;K. Kimata;Y. Okahata
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文献类型：
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作者：
Toshiaki Mori;T. Kodera;Hiroshi Yoshimine;Y. Kakuta;N. Sugiura;K. Kimata;Y. Okahata
利用高灵敏度的流动式27 MHz石英晶体微天平，检测了大肠杆菌K4菌株软骨素聚合酶（K4 CP）催化葡萄糖醛酸（GlcA）和N-乙酰半乳糖胺（GalNAc）逐步交替单糖转移至受体过程中的质量变化，并分析了K4 CP的延伸机制。发现K4 CP与软骨素受体强烈结合（K（d）=0.97 μM）。尽管各单体的结合亲和力和催化速率常数有很大差异，但表观催化效率（k（cat）/K（m））相似（GlcA转移为6.3×10（4）M（-1）s（-1），GalNAc转移为3.4×10（4）M（-1）s（-1））。这对于受体上GlcA和GalNAc的平滑交替延伸是合理的。这是第一个研究报告的酶，交替，糖伸长的动力学参数的测定。
Using a highly sensitive flow-type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one-sugar transfer of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K(d)=0.97 μM). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k(cat)/K(m)) was similar (6.3×10(4) M(-1) s(-1) for GlcA transfer and 3.4×10(4) M(-1) s(-1) for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.