1. The velocity of hydrolysis of gelatin by trypsin increases more slowly than the gelatin concentration and finally becomes nearly independent of the gelatin concentration. The relative velocity of hydrolysis of any two substrate concentrations is independent of the quantity of enzyme used to make the comparison.
2. The rate of hydrolysis is independent of the viscosity of the solution.
3. The percentage retardation of the rate of hydrolysis by inhibiting substances, is independent of the substrate concentration.
4. There is experimental evidence that the enzyme and inhibiting substance are combined to form a widely dissociated compound.
5. If the substrate were also combined with the enzyme, an increase in the substrate concentration should affect the equilibrium between the enzyme and the inhibiting substance. This is not the case.
6. The rate of digestion of a mixture of casein and gelatin is equal to the sum of the rates of hydrolysis of the two substances alone, as it should be if the rate is proportional to the concentration of free enzyme. This contradicts the saturation hypothesis.
7. If the reaction is followed by determining directly the change in the substrate concentration, it is found that this change agrees with the law of mass action; i.e., the rate of digestion is proportional to the substrate concentration.