Thiol and amino analogues as alternate substrates for glycerokinase from Candida mycoderma.
Thiol and amino analogues as alternate substrates for glycerokinase from Candida mycoderma.
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硫醇和氨基类似物作为来自念珠菌的甘油激酶的替代底物。
DOI：
10.1021/bi00440a005
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Cleland,WW
中科院分区：
文献类型：
--
作者：
Knight,WB;Cleland,WW
材料和方法材料。用KOH将缓冲液滴定至所需pH。(/?, 5）-1-巯基-2，3-丙二醇和（7 R，当用作底物时，来自Sigma和Aldrich的（S）-1-氨基-2，3-丙二醇显示可能由污染的甘油引起的突释。来自Aldrich和Sigma的硫醇分别含有0.7%和2%，而来自Aldrich的胺含有0.5%的快速反应物质。将这些化合物与甘油激酶、MgATP、丙酮酸激酶和磷酸烯醇丙酮酸在pH 7.5下孵育，然后进行Amicon过滤，得到无污染的产物。（R，S）-以及（R）-和（S）-2，2-二甲基-1，3-二氧戊环来自Aldrich。AMPPCP、AMPPNP、NAD和NADH来自Boeh-ringer。来自假丝酵母菌或土杆菌的甘油激酶来自Sigma，并在使用前相对于20 mM Hepes、5 mM二硫苏糖醇和0.3 mM EDTA透析，随后多次更换20 mM Hepes和5 mM二硫苏糖醇，并在氮气下储存。其他偶联酶和高纯度蔗糖均来自Sigma公司。在尼科莱NT-200或Bruker AM 500 NMR光谱仪上获得31 P和13 C NMR光谱，而在NT-200或Bruker WH 270仪器上获得质子NMR光谱。31 P NMR化学位移参考外部200 mM D3 PO 4，而质子和！3C化学位移参考外部TMS。安替林合成。以甘油为原料，经对甲苯磺酰化反应合成了（R，S）-1-巯基丙二醇1-磷酸酯，
Materials and Methods Materials. Buffers were titrated to the desired pH with KOH.(/?, 5)-l-Mercapto-2, 3-propandiol and (7?, S)-1-amino-2, 3-propanediol from Sigma and Aldrich when used as substrates displayed a burst caused presumably by contami-nating glycerol. The thiol from Aldrich and Sigma contained 0.7% and 2%, respectively, while the amine from Aldrich contained 0.5% fast-reacting material. Incubation of these compounds with glycerokinase, MgATP, pyruvate kinase, and phosphoenolpyruvate at pH 7.5 followed by Amicon filtration yielded products free of contamination.(R, S)-as well as {R)-and (5)-2, 2-dimethyl-1, 3-dioxolane was from Aldrich. AMPPCP, AMPPNP, NAD, and NADH were from Boeh-ringer. Glycerokinase from Candida mycoderma or Escher-ichia coli was from Sigmaand was dialyzed vs 20 mM Hepes, 5 mM dithiothreitol, and 0.3 mM EDTA, followed byseveral changes of 20 mM Hepes and 5 mM dithiothreitol, prior to use, and was stored under nitrogen. All other coupling en-zymes and high-purity sucrose were from Sigma.Methods. 31P and 13C NMR spectra were obtained on either Nicolet NT-200 or Bruker AM500 NMR spectrometers while proton NMR spectra were obtained on the NT-200, or on a Bruker WH270 instrument. 31P NMR chemical shifts are referenced to external 200 mM D3P04, while proton and! 3C chemical shifts are referenced to external TMS. Analogue Syntheses.(R, S)-1-Mercaptopropanediol 1-phosphate was synthesized from glycerol by tosylation and