Kumar Ashutosh, Chatterjee Sambarta, Nandi Mintu, Dua Arti
Department of Chemistry, Indian Institute of Technology, Madras, Chennai 600036, India.
J Chem Phys. 2016 Aug 28;145(8):085103. doi: 10.1063/1.4961540.
Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.
单体酶中的动态协同性以非米氏动力学行为为特征。后者被认为与包括由于酶构象波动导致的多条反应途径的机制有关。单分子荧光光谱学的最新进展为波动酶催化反应的潜在机制提供了新的基本见解。在这里，我们提出一种自下而上的方法，以从单分子随机轨迹中提取的机制为依据，来理解生理相关介观浓度下的酶周转动力学。这里提出的随机方法表明，米氏动力学的减慢导致负协同性，从而出现动态协同性。对于较少的酶，由于酶构象波动和分子离散性的综合作用而出现动态协同性。然而，酶数量的增加抑制了酶构象波动的影响，使得动态协同性仅由于反应物种数量的离散变化而出现。这些结果证实，基于平行途径米氏机制的波动酶的周转动力学随着酶数量的增加而转变为单途径米氏机制。对于大量的酶，随着随机动力学接近确定性行为，在非常高的底物浓度极限下会收敛到精确的米氏方程。
