Preparation and kinetic properties of a new form of chymotrypsin which is active at alkaline pH: α₁ chymotrypsin

The preparation of a new stable and active form of chymotrypsin is described. The enzyme possesses the threonine 147 instead of alanine 149 as the NH₂ terminal group of the C chain and has been called α₁ chymotrypsin. The conformational transition that affects chymotrypsins at alkaline pH was investigated and compared with that of α- and δ chymotrypsins by studying the kinetic constants and their pH dependencies for the hydrolysis of various specific ester substrates. The k(cat) values obtained with α₁ chymotrypsin are similar to those of α- and δ chymotrypsins. The K(m) values showed a progressive increase toward the alkaline pH region. The shape of the K(m) pH profiles closely resemble those of δ chymotrypsin and differ considerably from the behavior of α chymotrypsin. The results strongly implicate the participation of the alanine 149 amino group in the reversible inactivation of α chymotrypsin at high pH.