Unusual kinetic behavior of Endothia parasitica protease in hydrolysis of small peptides

Abstract

Endothia parasitica protease hydrolyzes l-leucyl- l-leucine amide and l-leucyl- l-phenylalanine amide at the peptide bond. l-Phenylalanyl- l-leticine amide, N-carbobenzoxy- l-leucyl- l-phenylalanine amide, N-carbobenzoxy- l-leucyl- l-pheml-alanine, N-carbobenzoxy- l-phenylalanyl- l-valine amide, and l-leucyl-β-naphthyl-amide are not hydrolyzed. In contrast to the kinetics of hydrolysis of casein and oxidized B-chain of insulin and activation of trypsinogen by Endothia parasitica protease which are normal, reaction progress curves for hydrolysis of l-leucyl- l-leucine amide and l-leucyl- l-phenylalanine amide are sigrnoidal. Initially, the reaction rates were of the order of 0.5–2.5% of the maximum rates eventually attained. With increasing time of incubation the reaction rates became faster and faster until maximum rates were achieved. This abnormal behavior was not eliminated by recrystallization of substrate or by incubation of enzyme alone or with products of the reaction prior to addition of substrate. Addition of a new aliquot of substrate, viz l-leucyl- l-leucine amide, to the reaction prior to complete hydrolysis of all of a previous aliquot of the same substrate, or reactions containing a mixture of oxidized B chain of insulin and l-leucyl- l-leucine amide, gave normal reaction progress curves. The duration of abnormal behavior before a maximum rate was attained was a function of enzyme concentration and temperature but not of substrate concentration even though substrate was in less than saturating amounts. The reaction data follow second-order autocatalytic kinetics with respect to enzyme concentration. It is proposed that most of the enzyme is in an inactive form in absence of substrate but is rapidly converted to the active form on combination with a good substrate such as trypsinogen, casein, or oxidized B chain of insulin. However, with a poor substrate such as l-leucyl- l-leucine amide, conversion to active enzyme is mediated through formation of an active enzyme-inactive enzyme complex followed by combination with substrate and hydrolysis.