期刊
CHEMBIOCHEM
卷 19, 期 6, 页码 596-603出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201700587
关键词
carbohydrates; conformation analysis; enzyme catalysis; kinases; reaction mechanisms
资金
- Federal Ministry of Science, Research and Economy (BMWFW)
- Federal Ministry of Traffic, Innovation and Technology (BMVIT)
- Styrian Business Promotion Agency (SFG)
- Standortagentur Tirol
- Government of Lower Austria
- Business Agency Vienna through the Austrian COMET-Funding Program
Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro--d-glucopyranose) in the presence of Mg2+-ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10 degrees C and pH7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of -d-glucose-6-phosphate in a manner reminiscent of an inverting -glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20-50mm), the apparent binding of levoglucosan (K-m=180mm) and ATP (K-m=1.0mm), as well as the inhibition by ADP (K-i=0.45mm) and d-glucose-6-phosphate (IC50=56mm). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d-glucose-6-phosphate and ADP was observed under a broad range of conditions. 6-Phospho--d-glucopyranosyl fluoride and 6-phospho-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol (6-phospho-d-glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip (C1C4)-C-4-C-1 required for 1,6-anhydro-product synthesis from d-glucose-6-phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据