FACILE ENZYMATIC DENOVO SYNTHESIS AND NMR SPECTROSCOPIC CHARACTERIZATION OF D-TAGATOSE 1,6-BISPHOSPHATE
FACILE ENZYMATIC DENOVO SYNTHESIS AND NMR SPECTROSCOPIC CHARACTERIZATION OF D-TAGATOSE 1,6-BISPHOSPHATE
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DOI：
10.1016/0008-6215(93)87020-s
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发表时间：
1993-01-15
影响因子：
3.1
通讯作者：
FESSNER, WD
中科院分区：
文献类型：
--
作者：
EYRISCH, O;SINERIUS, G;FESSNER, WD
从E.在半乳糖醇上生长的大肠杆菌细胞。的醛缩酶，一个同源四聚体组成的亚基的分子量约28000，具有最佳pH值为7.5，是高度选择性的L-β-D-苏型立体化学相比（99：1）。这允许其与甘油激酶、丙酮酸激酶和磷酸丙糖异构酶一起应用于偶联酶系统中，用于从二羟基丙酮和磷酸烯醇丙酮酸（用于原位再生腺苷三磷酸）开始以10 mmol的量从头一锅合成D-塔格糖1，6-二磷酸。即使在本工作期间对后者进行了改进之后，该快速方法在简单性和产率（总体为40%）方面与已知的多步化学制备相比也非常有利。从D-半乳糖醛酸开始的经典序列在两个磷酸化步骤中进行了修饰：通过应用三价亚磷酸化试剂二苄基二-N-乙基-亚磷酰胺酯化1，2：3，4-二-O-异亚丙基-D-塔格呋喃糖，然后过氧化氢氧化，并使用细菌果糖6-磷酸激酶进行D-塔格糖6-磷酸的酶促磷酸化。后者为重组大肠杆菌Pfk-2同工酶。coli中，测定了动力学常数。核磁共振波谱分配的D-塔格糖及其磷酸盐。
A D-tagatose 1,6-bisphosphate aldolase requiring Zn2+ for catalytic activity (class II) was purified from E. coli cells grown on galactitol. The aldolase, a homotetramer composed of subunits of mol wt approximately 28000, had a pH optimum at 7.5 and was highly selective for L-erythro as compared to D-threo stereochemistry (99: 1). This allowed its application in a coupled enzyme system together with glycerol kinase, pyruvate kinase and triose phosphate isomerase for the de novo, one-pot synthesis Of D-tagatose 1,6-bisphosphate starting from dihydroxyacetone and phosphoenolpyruvate (for the in situ regeneration of adenosine triphosphate), in quantities of 10 mmol. The expeditious process compares very favorably in simplicity and yield (40% overall) with the known multistep chemical preparation even after improvements to the latter accomplished during the present work. The classical sequence, which starts froM D-galacturonic acid, was modified at both phosphorylation steps: 1,2:3,4-di-O-isopropylidene-D-tagatofuranose was esterified by application of the trivalent phosphitylation agent dibenzyl di-N-ethyl-phosphoramidite followed by hydrogen peroxide oxidation, and a bacterial fructose 6-phosphate kinase was used for enzymic phosphorylation Of D-tagatose 6-phosphate. For the latter enzyme, which was the isoenzyme Pfk-2 from a recombinant strain of E. coli, kinetic constants were determined. NMR spectroscopic assignments are presented for D-tagatose and its phosphates.