Department of Physical Chemistry, University of Barcelona (UB), Barcelona, Spain.
J Phys Chem B. 2011 Feb 10;115(5):1115-21. doi: 10.1021/jp105296c. Epub 2010 Dec 29.
Traditionally, studies on the diffusion-controlled reaction of biological macromolecules have been carried out in dilute solutions (in vitro). However, in an intracellular environment (in vivo), there is a high concentration of macromolecules, which results in nonspecific interactions (macromolecular crowding). This affects the kinetics and thermodynamics of the reactions that occur in these systems. In this paper, we study the crowding effect of large macromolecules on the reaction rates of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by α-chymotrypsin, by adding dextrans of various molecular weights to the reaction solutions. The results indicate that the volume occupied by the crowding agent, but not its size, plays an important role in the rate of this reaction. A v(max) decay and a K(m) increase were obtained when the dextran concentration in the sample was increased. The increase in K(m) can be attributed to the slowing of protein diffusion, due to the presence of crowding. Whereas the decrease in v(max) could be explained by the effect of mixed inhibition by product, which is enhanced in crowded media. As far as we know, this is the first reported experiment on the crowding effect in an enzymatic reaction with a mixed inhibition by product.
传统上，生物大分子扩散控制反应的研究是在稀溶液（体外）中进行的。然而，在细胞内环境（体内）中，大分子的浓度很高，这导致了非特异性相互作用（大分子拥挤）。这会影响这些系统中发生的反应的动力学和热力学。在本文中，我们通过向反应溶液中添加各种分子量的葡聚糖来研究大分子拥挤对α-糜蛋白酶催化的 N-琥珀酰-L-苯丙氨酸-p-硝基苯胺水解反应速率的影响。结果表明，拥挤剂所占据的体积，而不是其大小，对该反应的速率起着重要作用。当样品中的葡聚糖浓度增加时，得到了 v(max)衰减和 K(m)增加。K(m)的增加可以归因于由于存在拥挤，蛋白质扩散的减慢。而 v(max)的降低可以用产物的混合抑制来解释，在拥挤的介质中，这种抑制作用增强。据我们所知，这是第一个关于具有产物混合抑制的酶反应中拥挤效应的报道实验。
