Effect of crowding by Dextrans in enzymatic reactions

Abstract

The interior of the living cell is highly concentrated and structured with molecules that have different shapes and sizes. Almost all experimental biochemical data have been obtained working in dilute solutions, situations which do not reflect the in vivo conditions. The consequences of such crowding upon enzymatic reactions remain unclear. In this paper, we have studied and compared the initial velocity of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by alpha-chymotrypsin, the oxidation of ABTS by H2O2 catalyzed by HRP and the oxidation of NADH in presence of pyruvate catalyzed by LDH. These reactions were chosen as model enzymatic processes occurring in different in vitro crowded media. The systems crowding has been built by introducing Dextran of several concentrations and sizes. Our results indicate that the volume occupied by the crowding agent, but not its size, plays an important role on the initial velocity of reactions involving tiny enzymes. However, the enzyme size is another important factor influencing the velocity of the reactions of large enzymes occurring in Dextran crowded media. In this situation, the reaction initial velocity depends on both occupied volume and dimension of the crowding agent that is present in the reaction media.

Keywords: Alpha-chymotrypsin; Dextran; Enzyme kinetics; HRP; LDH; Macromolecular crowding.