Effect of Crowding by Dextrans on the Hydrolysis of N-Succinyl-l-phenyl-Ala-p-nitroanilide Catalyzed by α-Chymotrypsin

Abstract

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 non-specific 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 Nsuccinyl-L-phenyl-Ala-p-nitroanilide catalyzed by alpha-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.

Macromolecular Crowding Effect upon in Vitro Enzyme Kinetics: Mixed Activation–Diffusion Control of the Oxidation of NADH by Pyruvate Catalyzed by Lactate Dehydrogenase

“…They showed that for samples of Dextrans of about 20% (w/w) the reaction rates decrease with the increase of Dextran size: a decrease of about 2, 5 and 7-fold was observed for Dextrans of 15-70, 200 and 500 kDa, respectively However, it is in contrast with the results showed in our previous works 37,41 and with the results of Minton et al 2,[8][9][10][11][12] . In all these works, only excluded volume but not obstacle size was presented as a major factor influencing enzymatic reactions occurring in reaction media crowded by macromolecules.…”

“…In a situation of diffusion control, an increase in ‫ܭ‬ ୫ value can be expected since the bimolecular constant (݇ ଵ ) will decrease with the crowding [45][46][47] . In the literature, there are few works that report this situation 21,37,[45][46][47] . In these reported cases, a high diffusion resistance in the sample is responsible of a ‫ܭ‬ ୫ increase with Dextran concentration.…”

Monte Carlo simulations of enzymatic reactions in crowded media. Effect of the enzyme-obstacle relative size

“…In contrast, to account for the usual greater size of the crowding molecules and to analyse the effect of the relative crowder size in the diffusion-reaction process, four different obstacles sizes have been considered (see Fig. 1 Then, for a typical enzymatic reaction [33], with…”

“…However, after the pioneering work of Laurent in 1971 [21], quite a few studies have explored the effects of crowding on enzyme catalysis, even in vitro [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39].…”

“…In recent years, the effects of crowding on enzyme catalysis have been explored by different experimental works, excellently depicted by Zhou et al [16] and Noris and Malys [30]. Some of the studies present distinct effects produced by various crowding agents on the enzymes kinetics [26,33,[38][39]66]. These effects are given by different experimental conditions considering the type, shape, size and excluded volume of the used crowders.…”