THE KINETICS OF TRYPSIN DIGESTION : II. CONDITIONS UNDER WHICH THE REACTION IS MONOMOLECULAR.

1. The kinetics of enzyme reactions diverge more or less from the simple mass action expression for a monomolecular reaction. There is good reason to believe that these discrepancies are due to other secondary reactions which also agree with the law of mass action. Attempts to incorporate all these reactions in one equation, however, are unsatisfactory owing to the complexity of the relations involved. 2. It is possible, however, to regulate conditions experimentally so that these secondary reactions are reduced to a minimum. This has been done in the case of trypsin digestion by working at a low temperature, which prevents inactivation of the trypsin, by using a large amount of trypsin, which prevents the inhibiting effect of the products from becoming noticeable, and by using the disappearance of the protein as the indicator which obviates the complicating effects of the consecutive reactions. 3. Under these conditions the hydrolysis, for any initial concentration of casein is accurately represented by the monomolecular formula, See PDF for Equation The effect of variations in the initial trypsin concentration are also correctly predicted. 4. If the initial casein concentration is varied, however, the value of the constant changes for each casein concentration, becoming less as the casein increases and eventually becoming nearly inversely proportional to the casein concentration. It is pointed out that this cannot be due to a compound between enzyme and casein, nor to the viscosity, but is probably owing to an equilibrium between the casein and water, in which the casein can be replaced by the first cleavage products. This is corroborated by the fact that if the casein is dissolved in a freshly prepared solution of digested casein, the anomalous effect of the substrate concentration disappears and the reaction is typically monomolecular in every respect. A solution of digested casein which has been in prolonged contact with trypsin does not have this effect. 5. It is pointed out that the various equations that have been proposed to account for the enzyme reactions on the basis of a compound between the enzyme and substrate could be applied equally well on the basis of a compound between water and the substrate which is attacked by the enzyme.