Thrall S H, Mehl A F, Carroll L J, Dunaway-Mariano D
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Biochemistry. 1993 Feb 23;32(7):1803-9. doi: 10.1021/bi00058a014.
The intermediacy of a pyrophosphorylenzyme (E-PP) and phosphorylenzyme (E-P) in the Clostridium symbiosum pyruvate phosphate dikinase catalyzed interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), inorganic pyrophosphate (PPi), and phosphoenolpyruvate (PEP) was examined using transient kinetic techniques. Single-turnover experiments with [gamma-32P]ATP or [14C]ATP and PPDK were carried out in the presence and absence of Pi to test for pyrophosphorylenzyme and AMP formation, respectively. Formation of the E-PP.AMP complex was found to be followed by Pi binding and the formation of the E-P.AMP.PPi complex. The level of pyrophosphorylenzyme accumulated during a single turnover was found to be dependent on the divalent metal cofactor used (Mn2+ > Co2+ > Mg2+). Single-turnover experiments with [32P]PEP and PPDK were carried out in the presence and absence of PPi and pyruvate to test for phosphorylenzyme formation in the reverse, ATP-forming direction of the reaction. Phosphorylenzyme formed from the reaction of the E.PEP complex was converted in the presence of AMP and PPi to free enzyme at a rate exceeding the steady-state turnover rate. The reaction sequence for pyruvate phosphate dikinase was determined to be [formula see text] 31P NMR analysis of the phosphorylenzyme in the native (-4.0 ppm) and denatured form (-3.9 ppm) revealed a 3-N-phosphohistidine residue. Complexation of Mg2+ resulted in a 0.3 ppm upfield shift of the phosphorus resonance from native phosphorylenzyme while Mn2+ complexation lead to extensive line broadening, indicative of metal cofactor binding in close vicinity to the phosphoryl group.(ABSTRACT TRUNCATED AT 250 WORDS)
利用瞬态动力学技术研究了共生梭菌丙酮酸磷酸二激酶催化的三磷酸腺苷（ATP）、正磷酸盐（Pi）和丙酮酸与一磷酸腺苷（AMP）、无机焦磷酸盐（PPi）和磷酸烯醇式丙酮酸（PEP）相互转化过程中焦磷酸化酶（E-PP）和磷酸化酶（E-P）的中间体情况。分别在有和没有Pi的情况下，用[γ-32P]ATP或[14C]ATP与丙酮酸磷酸二激酶进行单周转实验，以检测焦磷酸化酶和AMP的形成。发现E-PP.AMP复合物的形成之后是Pi的结合以及E-P.AMP.PPi复合物的形成。发现在单周转过程中积累的焦磷酸化酶水平取决于所使用的二价金属辅因子（Mn2+＞Co2+＞Mg2+）。在有和没有PPi及丙酮酸的情况下，用[32P]PEP与丙酮酸磷酸二激酶进行单周转实验，以检测反应逆向生成ATP方向上磷酸化酶的形成。由E.PEP复合物反应形成的磷酸化酶在AMP和PPi存在下以超过稳态周转速率的速度转化为游离酶。确定丙酮酸磷酸二激酶的反应序列为[公式见原文]。对天然形式（-4.0 ppm）和变性形式（-3.9 ppm）的磷酸化酶进行的31P NMR分析揭示了一个3-N-磷酸组氨酸残基。Mg2+的络合导致天然磷酸化酶的磷共振信号向上场移动0.3 ppm，而Mn2+的络合导致谱线大幅展宽，表明金属辅因子在靠近磷酰基的位置结合。（摘要截短于250词）
