ON SPONTANEOUS AND ALPHA-CHYMOTRYPSIN-CATALYZED HYDROLYSIS OF 4-CIS-BENZYLIDENE-2-PHENYLOXAZOLIN-5-ONE . CATALYSIS BY TRIS(HYDROXYMETHYL) AMINOMETHANE

4-cis-Benzylidene-2-phenyloxazolin-5-one and N-acetyl-L-valine p-nitrophenyl ester have been considered as substrates for α-chymotrypsin. Results obtained indicate the desirability of activated derivatives of TV-acylamino acids as substrates in specificity studies. The reactions of Tris with 4-cis-benzylidene-2-phenyloxazolin-5-one and the corresponding acylenzyme, α-benzamido-cis-cinnamoyl-α-chymotrypsin, have been investigated. In both cases, the Tris amide of α-benzamido-cis-cinnamic acid (λmax 282 mμ) is the final product, and reaction occurs through an intermediate species (λmax 285 mμ). This intermediate is relatively stable, and was isolated and examined. Evidence obtained suggests that Tris intervention in these systems produces the Tris ester of α-benzamido-cis-cinnamic acid as the intermediate species. This ester is subsequently converted to the Tris amide in both the enzymatic and nonenzymatic systems. © 1969, American Chemical Society. All rights reserved.