Activation of human leukocyte elastase activity by excess substrate, hydrophobic solvents, and ionic strength.

The human leukocyte elastase-catalyzed hydrolysis of N-succinyl-(L-alanine),-p-nitroanifides (n = 2 to 5) is activated at high substrate concentrations. For each substrate, four kinetic parameters have been determined: kcat and K,,,, the constants extrapolated from data obtained at low substrate concentrations; Kcat, the turnover number at infinite substrate concentration; and IC,,,, the dissociation constant of the second substrate molecule acting as an activator. The mixture of leukocyte elastase isoenzymes yields the same constants with N-succinyl-(L-alanine)3-p-nitroanilide as the major isoenzyme E4 or the mixture of the minor isoenzymes Ez + Ea, i e . kcat = 0.15 s-‘, K,,, = 0.5 mM, Kcat = 0.7 sal, and IC,,, = 15 mM. K,,, decreases and kcat increases when n increases from 2 to 4, whereas addition of 1 more alanine residue has an unfavorable effect on both constants. Substrate activation is also observed with the stable complex formed between human plasma az-macroglobulin and leukocyte elastase. The macroglobulin increases both kcat and VCat values. The elastase-catalyzed hydrolysis of N-succinyl-(L-alanine)3-p-nitroanilide is also accelerated by a series of organic solvents and by ionic strength. Except for Nmethylpyrrolidone and tetrahydrofuran, this activating effect is directly related to the hydrophobicity of the solvents. Dimethylformamide (10% v/v) increases both kcat (6.2 times) and K,,, (10 times) and abolishes the phenomenon of substrate activation. By contrast, activation by excess substrate still occurs at high ionic strength: in the presence of 2 M NaCl, both kcat and Kcat are increased by a factor of 5, K,,, increases by a factor of about 3, and IC, remains essentially unchanged. Ionic strength and organic solvents do not compete for the activation of elastase catalysis. Determination of the kinetic constants of the elastase-catalyzed hydrolysis of N-succinyl-(~-alanine)a-pnitrophenyl ester shows that acylation is the rate-limiting step for the hydrolysis of the corresponding anilide substrate. Excess substrate, organic solvents, and ionic strength increase the rate of formation of the acyl-enzyme. On the other hand, with the ester substrate, for which deacylation is rate-limiting, no substrate activation could be observed and the organic solvents had a slightly inhibitory effect.