Conformation and Activity of Chymotrypsin: The p H-Dependent, Substrate-Induced Proton Uptake

Abstract

Hydrogen ion uptake by chymotrypsin during reversible binding of specific substrate is shown to be due to an ionizing group of the enzyme with a pK(apparent) approximately 9 in the free enzyme. This pK(apparent) is shifted to higher value in the enzyme-substrate complexes. Previous results indicating an equilibrium, controlled by this ionizing group, between active and inactive conformational forms of chymotrypsin are confirmed.

Binding of competitive inhibitors to δ-chymotrypsin in the alkaline pH region. Competitive inhibition kinetics and proton-uptake measurements

“…This situation contrasts with the sigmoidal profile with a maximal proton uptake of one proton (per mole of enzyme) found previously with -chymotrypsin as expected for a pA'a shift of the order of 2.0 or more pATa units (Wedler and Bender, 1969). Qualitatively similar differences between the enzymes have been reported by McConn et al (1968) who studied the proton uptake upon binding of vV-acetyl-D-trvptophanamide and /V-acetyl-L-tryptophanamide to -chymotrypsin and -chymotrypsin, respectively. Our proton uptake data on -chymotrypsin also agree with the results reported recently by Garel and Labouesse (1970) who used indole as a competitive inhibitor.…”

“…Equation 3 was used in the calculation of the theoretical 1 Similar schemes to that of eq 2 have been used previously to account for pH dependence data of chymotrypsin Kaplan and Laidler, 1967;McConn et al, 1968;Garel and Labouesse, 1970).…”

“…We have measured the pH dependence of the binding constants of the competitive inhibitors benzyl alcohol, tryptophol, and Nacetyl-D-tryptophanamide by the use of competitive inhibition kinetics. As recent studies indicate that the binding of substrates and competitive inhibitors to a-and -chymotrypsin causes an uptake of protons from the medium (Bender and Wedler, 1967;McConn et al, 1968; Wedler and Bender, 1969;Garel and Labouesse, 1970), we have also measured the pH dependence of the proton uptake observed upon binding of the above three inhibitors to -chymotrypsin.…”

X-Ray Diffraction Studies of Enzymes

“…The blocking of this a-amino group by acetylation destroys activity, and the attachment of irreversible inhibitors to the active site greatly increases its pKa ( 135,136) . Substrate binding is reduced at high pH (137), and the presence of substrate perturbs the pKa of Ile 16 by stabilizing the active conformation (138,139).…”

Solvolytic reactions of endo-bicyclo[3.2.1]oct-6-en-8-yl tosylate

“….OTs X 4 Acid hydrolysis of the ketal of bicyclo[4.2.0]oct-7-en-2-one (5)6 gave the known ketone, bicyclo[4.2.0]oct-7en-2-one (6).7 Reduction of 6 with lithium aluminum hydride gave a mixture of the corresponding epimeric alcohols (7). 8 The tosylates of 7 were prepared and subjected to buffered acetolysis.…”