The behavior of α-chymotrypsin has been studied in the simultaneous presence of two different substrates, each present in the reaction mixture at its saturation level. Mixtures of two esters were hydrolyzed at rates intermediate between the rates of hydrolysis of each ester when present alone, suggesting, in this case, competitive hydrolysis. In contrast, the rates of hydrolysis in mixtures of casein with gelatin or of either protein with an ester were equal to the sum of the rates of hydrolysis of the separate substrates, indicating in these cases independent hydrolysis. The activity of the α-chymotrypsin preparation used could not be attributed to contamination with other enzymes. Studies of the effect of soy bean inhibitor on chymotrypsin indicate that the mechanism of inhibition with protein substrates differs from that when esters are used, providing further evidence that α-chymotrypsin reacts differently with esters and proteins.
These results indicate that if chymotrypsin forms specific complexes with its substrates, it must possess at least three distinct active sites. However there is independent chemical evidence that the proteolytic and esterolytic activities of this enzyme reside in the same active center. If this is true, the experimental observations reported here cannot be explained unless it is supposed that this enzyme does not form specific Michaelis complexes with its substrates.