Studies of the activity of chymotrypsin

Abstract

Physical and chemical studies of chymotrypsin in solution and kinetic measurements of individual steps in reactions catalysed by this hydrolytic enzyme are reported. The solution studies also provide a basis for correlation of individual reaction steps with structural information obtained by Drs Blow and Hartley and discussed at this Discussion. The rate and equilibrium constants pertaining to the three-step mechanism shown below have been determined for the chymotrypsin catalysed hydrolysis of three specific ester substrates—the ethyl esters of V-acetyl-L-tyrosine, N -acetyl-L-tryptophan, and N -acetyl-L-phenylalanine—at selected pH values with use of both flow and relaxation techniques. iV-acetyl-L-phenylalanine—at selected pH values with use of both flow and relaxation techniques. / * E + S ^ 4 E S -y » EP 2 ,---► E + P 2 , * 2 3 £ 3 4 where E is enzyme, S is substrate, ES is a complex, EP 2 is an intermediate compound, and the products P 1 and P 2 are, respectively, an alcohol or amine and an acid. The hydrolysis of chymotrypsin specific substrates is shown to be considerably more complex than is indicated by this previously proposed mechanism. In addition to the steps shown, there exist a number of ionization and conformational equilibria which account for the bell-shaped pH-rate profiles observed for chymotrypsin catalysed reactions. A catalytically active enzyme conformation, which is in pH dependent equilibrium with a catalytically inactive conformation, is shown to have its origin in the enzyme activation mechanism. In the chymotrypsin catalysed hydrolysis of an amide substrate, furylacryloyltryptophanamide, there has been detected an additional intermediate which differs from the intermediate EP 2 observed in ester hydrolysis.

The Conformational Oscillation of delta-Chymotrypsin Involvement of Methionine-192

“…Fig. 3 shows that the molar ellipticity is similar for the free and for the oxidized enzyme at neutral pH and at a pH which is way above the isoleucine-16-dependent alkaline transition [1,2]. The major change observed between pH 7 and pH 11 with the free enzyme is retained with the methionine-192-oxidized one.…”

“…7 indicates and one may conclude that in the alkaline structure of 6-chymotrypsin methionine-192 presents little or no chemical reactivity towards iodoacetic acid. This reactivity increases with a lowering of the initial pH of the enzyme according to a dependence that is exactly that of the alkaline transition of 6-chymotrypsin [1,2,33]. This can be interpreted as evidence that methionine-192 moves from one position where it has no reactivity to a new one where it is reactive while the protein isomerizes from its alkaline to its neutral structure.…”

“…a) It is dependent on a single group of apparent pK 9 at 15 "C (or 8.3 at 25 "C) which is that of isoleucine-16 [1,2,8,28,30].…”

The Time Dependence of the Activity of δ‐Chymotrypsin at High pH

“…Chymotrypsin indeed shows catalytic activity up to pH 11 or more [13]. At high pH, non-covalent binding of a specific inhibitor shifts the enzyme conformation from the E' to the E state [6,11] and this displacement of the E + E ' equilibrium is admitted to account for the p H dependence of chymotrypsin activity in the alkaline pH range [6,11].…”

“…Chymotrypsin indeed shows catalytic activity up to pH 11 or more [13]. At high pH, non-covalent binding of a specific inhibitor shifts the enzyme conformation from the E' to the E state [6,11] and this displacement of the E + E ' equilibrium is admitted to account for the p H dependence of chymotrypsin activity in the alkaline pH range [6,11].The isomerization between the E and E' conformations is a slow process [7,17] that does not seem to be greatly accelerated by the presence of ligands [lo, 12,18,19]. The rate at which this isomerization occurs is much lower than that of any step of the chymotrypsin-catalyzed hydrolysis of a specific ester substrate [20].…”