The Active Site in α-Chymotrypsin

The active site of α-chymotrypsin is considered to be composed of parts complementary to the parts of a small molecule natural substrate, l-ethyl N-acetyl β-phenylalaninate, in a conformation in which the β-aryl and hydrolyzing ester groups are transoid: (a) the ar site, a cavity or fold into the enzyme at which the β-aryl substituent provides the primary associative effect; (b) the am site, at or near the enzyme surface, at which the α-acylamido group associates by hydrogen bonding; (c) the h site of restricted volume into which the α-hydrogen atom or other small substituent may fit; (d) the hydrolytic site, n, at or near the enzyme surface, at which the group to be hydrolyzed must be placed if an association is to be effective. High reactivity of noncyclized substrates requires binding at am and ar which restricts rotation about Cα—Cβ and Cβ—Cphenyl (Cφ), and a fit at h, placing the group to be hydrolyzed at n. d Enantiomers hydrolyze (a) when the α substituents associate at ar, and β substituents fit at am or are absent, and (b) when the α substituent may fit at h. Effective association and hydrolysis of d enantiomers lead to absence of or inversion of stereospecificity. dl-Methyl 3,4-dihydroisocoumarin-3-carboxylate (I) [see PDF for structure] has been prepared. It is hydrolyzed rapidly by α-chymotrypsin, leading to l(+)-unhydrolyzed Ester I, and d(-)-Acid VI. The acid l(+)-VI was prepared, stereospecifically, from [see PDF for structure] l(-)-phenyllactic acid, establishing the preferred enzymic hydrolysis of d(-)-I. A convenient stereospecific synthesis of d(-)-1-keto-3-carbomethoxytetrahydroisoquinoline (II) [see PDF for structure] from d(+)-β-phenylalanine has also been developed. Kinetic parameters for hydrolysis by α-chymotrypsin are: d(-)-I, kcat = 42 sec-1, Km = 0.0007 m, kcat:Km = 6.0 x 104 m-1 sec-1; l(+)-I, kcat = 1.8 sec-1, Km = 0.028 m, kcat:Km = 64 m-1 sec-1. The cyclic ester group of d(-)-I may not utilize the am site effectively, and it is concluded that the cyclic amide group of d(-)-II also does not utilize the am site. The α-heteroatoms of d(-)-I and d(-)-II fit at h, the aryl groups at ar, leading to hydrolysis of the d enantiomers. The heterocyclic rings of d(-)-I and d(-)-II lead to high reactivity by restricting rotations at Cα—Cβ and Cβ—Cφ. This, coupled with association of the phenyl ring at ar, places the carbalkoxyl group at n. The lesser reactivity of l-cyclized substrates appears related to diminished access of the aryl group to the ar site when the cyclic amide or ester group in II and I associates or lies at am. The proposal that the carbalkoxyl group in d(-)-I and d(-)-II is more nearly equatorial in the reacting conformation is discussed and supported. The usefulness of cyclized substrates with respect to information about the ar and h sites and their limitation with respect to the am site are indicated.