Temperature and pH dependence of the association rate constant of elastase with alpha 2-macroglobulin.

Binding kinetics of porcine pancreatic elastase to human alpha 2-macroglobulin was monitored by measuring the enzymatic activity of alpha 2-macroglobulin-bound elastase on succinyltrialanine p-nitroanilide after inhibition of free elastase by alpha 1-proteinase inhibitor. The association of the two proteins follows second-order kinetics with a rate constant ka = 4.4 X 10(6) M-1 S-1 at pH 8.0 and 25 degrees C. The rate of association strongly increases between pH 5.0 and 8.0, suggesting that the rate-limiting step of binding is the proteolytic cleavage at the bait region of the macroglobulin. The study of the temperature dependence of ka shows that the binding of elastase to alpha 2-macroglobulin is characterized by a positive entropy of activation (delta S* = +35.3 e.u. at 25 degrees C).