重组丙酮酸磷酸双激酶与荧光素酶偶联催化ATP-AMP循环反应

丙酮酸磷酸双激酶（pyruvate phosphate dikinase, PPDK; EC 2.7.9.1）能够可逆催化磷酸烯醇式丙酮酸（phosphoenolpyruvate, PEP）、单磷酸腺苷（adenosine monophosphate, AMP）和焦磷酸盐（pyrophosphate, PPi）生成三磷酸腺苷（adenosine triphosphate, ATP）、无机磷酸盐（orthophosphate, Pi）和丙酮酸（pyruvate）.以热玫瑰小双孢菌基因组DNA为模板，PCR扩增得到了编码PPDK的基因，将此基因片段插入表达载体pET24a (+)，在大肠杆菌中表达C端融合His-Tag的重组PPDK.与我们先前表达的N端融合His-Tag的PPDK相比，酶的活性提高了20倍，提示该酶的N端对活性十分重要.重组PPDK单体分子量为98 kD.经过镍亲和层析和超滤后，重组PPDK基本达到电泳纯.重组PPDK与荧光素酶偶联能够形成1个ATP-AMP循环反应，在该循环反应中，荧光素酶催化ATP生成的AMP和PPi能够被PPDK重新转化成ATP，产生一个持续稳定的信号.

An Enzymatic Cycling Reaction for ATP and AMP with Concomitant Use of Firefly Luciferase and Recombinant Pyruvate Phosphate Dikinase

Pyruvate phosphate dikinase (PPDK; EC 2.7.9.1) is found in certain microorganisms and plants, and catalyzes the conversion of AMP, PPi and phosphoenolpyruvate (PEP) to ATP, Pi and pyruvate. Using the genomic DNA of Microbispora rosea subsp.aerata as the template, a DNA fragment encoding the gene PPDK was amplified by PCR and inserted into the expression vector pET24a (+). We expressed PPDK fused to a C-terminal sequence of His-Tag in Escherichia coli and obtained an enzymatically active protein, whose specific activity was 20-fold higher than that of PPDK fused to a N-terminal sequence of His-Tag expressed previously. This result indicated that N-terminal sequence of PPDK takes an important role in activity containing. The molecular weight of PPDK was estimated to be 98 kDa by SDS-PAGE. The optimal temperature and optimal pH for PPDK-catalyzed reaction are 50 ℃ and 7.0, respectively. PPDK was stable in the pH range from 7 to 10, and retained 80% activity after heat-treatment at 50 ℃ for 60 min. PPDK was purified by His?Bind Resin affinity chromatography and ultrafiltration using 10-kDa cut-off membranes. A bioluminescent enzymatic cycling assay for ATP and AMP with concomitant use of firefly luciferase and PPDK was demonstrated. In this system, AMP and PPi produced from ATP by firefly luciferase were converted back into ATP by PPDK. This resulted in constant luminescence once the stable phase had been reached.