Stereochemistry of the active site of -chymotrypsin. The influence of polar groups in locked substrates.

Abstract The binding site of α-chymotrypsin (EC 3.4.4.5) has been "mapped" to identify areas displaying steric hindrance to binding and areas of polar environment. Of the five isomeric methyl benzindancarboxylates tested as substrates, only methyl-4,5-benzindan-2-carboxylate (Compound X) underwent chymotryptic hydrolysis with moderate stereospecificity. This finding confirms Niemann's hypothesis that the aromatic binding site in chymotrypsin is planar, elongated, and curved. Comparison of the reactivities of X and methyl indan-2-carboxylate with their oxygen analogues, methyl 1,2-dihydronaphtho[2,1-b]furancarboxylate and methyl hydrocoumarilate, reveals that the polar atoms or groups adjacent to the α-carbon in locked substrates do not contribute significantly to the over-all reactivity or binding of these substrates when they are oriented productively but that they are detrimental when located incorrectly. This concept is supported by the extremely slow rate of hydrolysis of methyl 2-acetamidoindan-2-carboxylate (Compound VIII), a new rigid analogue of methyl N-acetylphenylalaninate (Compound VI). This observation indicates that the site that accommodates the acetamido group of l-VI is not left unoccupied when reactive enantiomers of cyclized substrates undergo chymotryptic hydrolysis, but the interaction there is imperfect.