Wang H C, Ciskanik L, Dunaway-Mariano D, von der Saal W, Villafranca J J
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Biochemistry. 1988 Jan 26;27(2):625-33. doi: 10.1021/bi00402a020.
The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)
利用多种不同的动力学诊断方法研究了共生拟杆菌丙酮酸磷酸二激酶（PPDK）的动力学机制。对于AMP/PPi和ATP/Pi底物对，初始速度模式呈相交状，而对于所有其他底物对则呈平行状。结果表明，在没有共底物的情况下，PPDK能催化[14C]丙酮酸与磷酸烯醇丙酮酸（PEP）的平衡交换；在存在Pi/PPi但不存在它们时，能催化[14C]AMP与ATP的平衡交换；在存在ATP/AMP但不存在它们时，能催化[32P]Pi与PPi的平衡交换。通过使用[αβ-18O，β，β-18O2]ATP和[βγ-18O，γ，γ，γ-18O3]ATP以及31P NMR技术，还表明该酶能催化ATP中αβ桥氧与α-P非桥氧之间以及βγ桥氧与β-P非桥氧之间的交换。这些交换是在存在Pi但不存在Pi时由PPDK催化的。这些结果被解释为支持双（ATP，Pi）双（AMP，PPi）单（丙酮酸）单（PEP）机制。通过进行终产物抑制研究来考察AMP和Pi的结合顺序。发现终产物抑制剂5'-单磷酸硫代腺苷（AMPS）对AMP具有竞争性，对PPi无竞争性，对PEP无竞争性。发现终产物抑制剂亚氨基二磷酸（PNP）对PPi具有竞争性，对AMP无竞争性，对PEP无竞争性。这些结果表明AMP在PPi之前结合。通过进行抑制、位置同位素交换和替代底物研究来研究ATP和Pi的结合顺序。（摘要截于第250字）
