Nα-Benzoyl-L-arginine

Trypsin is an important proteolytic enzyme in the digestive system and its activity is a major indicator for evaluating diseases such as chronic pancreatitis. Here, we present a novel label-free method to detect trypsin kinetics using a nanopore technique. A mutant α-hemolysin (M113R)7 protein nanopore equipped with a polyamine decorated β-cyclodextrin (am7β-CD) was employed as a sensing platform for the real-time monitoring of the process of trypsin enzymatic cleavage of a substrate Nα-benzoyl-l-arginine ethyl ester (BAEE) at the single molecule level. Significantly, this sensor can exclusively respond to the current modulation caused by the product and prevent interference from the substrate, thus improving detection sensitivity, and it provides a new scheme to detect enzyme activity for cleaving small molecules.

The hydrolysis reaction of Nalpha-benzoyl-L-arginine ethyl ester catalyzed by trypsin from pig pancreas was comparatively studied in an aqueous buffer solution and in the system of reversed micelles of Aerosol OT in octane (pH 8.5) to determine the mechanisms of influence of the enzyme microenvironment on the rate constants of the elementary stages of the enzymatic reaction. The temperature dependences of the catalytic constant kcat and the rate constant of the second order kcat/Km (s, catalysis efficiency) allowed the determination of the rate constants and the activation energy of elementary stages of the enzymatic reaction. It was revealed that a decrease in the efficiency of catalytic action of trypsin in inverted mycelles in comparison with an aqueous solution is first of all determined by a decrease in the rate constant of formation of the enzyme-substrate complex k1. Possible mechanisms of the effect of the microenvironment on the elementary stages of catalytic action of the enzyme are discussed.

Isothermal titration calorimetry (ITC) is a universal technique that directly measures the heat absorbed or released in a process. ITC is typically used to determine thermodynamic parameters of association of molecules without the need to label them. However, ITC is still rarely applied to study chemical reactions catalyzed by enzymes. In addition, these few studies of enzyme kinetic measurements that have been performed were in diluted solutions. Yet, to estimate realistic kinetic parameters, we have to account for the fact that enzymatic reactions in cells occur in a crowded environment because cells contain 200-400 g/L of macromolecular crowders such as proteins, ribosomes and lipids. Thus we expanded the ITC application for solutions mimicking the cellular environment by adding various macromolecular crowders. We investigated how these crowders affect the kinetics of trypsin-catalyzed reactions and determined the Michaelis-Menten parameters for hydrolysis of two trypsin substrates: Nα-benzoyl-l-arginine ethyl ester (BAEE) and Nα-benzoyl-dl-arginine β-naphthylamide (BANA). Since ITC enables investigations of complex and turbid solutions with label-free reagents, it seems a perfect technique for kinetic analyses in crowded solutions. ITC also offers the opportunity to control enzyme-crowder and substrate-crowder interactions.