Cane D E, Chiu H T, Liang P H, Anderson K S
Department of Pharmacology, 333 Cedar Street, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Biochemistry. 1997 Jul 8;36(27):8332-9. doi: 10.1021/bi963018o.
The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods. The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C. In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat. These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site. Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100. The deuterium isotope effect on the consumption of [1-2H,1,2-14C]FPP was found to be 1.11 +/- 0.06. Single turnover reactions of [1,2-14C]FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP). However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay. Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway.
通过快速化学淬灭法研究了里氏木霉菌烯合酶反应的预稳态动力学。发现单轮反应速率为3.5 - 3.8 s⁻¹，该速率比在15℃下里氏木霉菌烯合酶催化法呢基二磷酸（FPP）转化为里氏木霉菌烯的稳态催化速率（kcat = 0.09 s⁻¹）快40倍。在多轮反应实验中，对应于里氏木霉菌烯在酶表面积累的爆发相（kb = 4.2 s⁻¹）之后是产物较慢的稳态释放（klin = 0.086 s⁻¹），这与kcat相对应。这些结果强烈表明，里氏木霉菌烯从酶活性位点的释放是整个反应中的限速步骤，而FPP的消耗是限制活性位点化学催化的步骤。对里氏木霉菌烯合酶突变体D101E进行的单轮反应实验表明，其稳态速率常数kcat是野生型的1/3，该突变实际上使FPP消耗速率降低了100倍。发现[1-²H,1,2-¹⁴C]FPP消耗的氘同位素效应为1.11±0.06。由里氏木霉菌烯合酶催化的[1,2-¹⁴C]FPP的单轮反应在4℃、15℃或30℃下进行，旨在直接观察所提出的中间体橙花叔基二磷酸（NPP）。然而，未检测到NPP，这表明NPP的转化一定太快以至于在该检测方法的检测限内无法观察到。综上所述，这些观察结果表明FPP异构化为NPP是限制里氏木霉菌烯合酶反应途径中化学催化速率的步骤。
