On the Active Site of α-Chymotrypsin ABSOLUTE CONFIGURATIONS AND KINETICS OF HYDROLYSIS OF CYCLIZED AND NONCYCLIZED SUBSTRATES

Abstract The absolute steric course, stereoselectivity, and kinetics of hydrolysis of cyclized and noncyclized ester substrates by α-chymotrypsin are compared. The cyclized substrates are of the form [see PDF for sequence] The noncyclized substrates are analogous α-substituted β-phenylpropionates, C6H5CH2CH(X)CO2R, II-A, X = —NHCOCH3; II-B, X = OCOCH3; II-C, X =—CH2CO2R; V-A, X = Cl; V-B, X = OH; V-C, X = CH3. The absolute configurations of I-C, I-F, and I-G are established in this work; absolute configurations of the other compounds were previously known or recently established. The active site of α-chymotrypsin is described in terms of loci complementary to a conformation of l(+)-II-A, in which the aryl and ester groups are transoid, the β-aryl and α-hydrogen are directed into the enzyme, and the α-acetamido and hydrolyzing ester groups are directed along the exterior of the enzyme. Conditions for hydrolysis of l and d enantiomers by α-chymotrypsin are described. Association is generally dominated by the β-aryl group at its locus, ar. Hydrolysis requires the ester group to be at the hydrolytic site n. Compounds II-A, II-B, and II-C hydrolyze in the l sense as the large α-substituents occupy the α-acetamido site, am, and may not fit into the α-hydrogen locus, h. Both enantiomers of V-A and V-B are hydrolyzed as the small polar substituents, Cl and HO, may be directed in the l sense toward am and in the d sense toward h. Only the l enantiomer of V-C is hydrolyzed, and the small hydrocarbon group, CH3, may not fit at h. All of the cyclized substrates, IA-IG, with five- and six-membered second rings, are hydrolyzed preferentially in the d sense, but with widely varying reactivity and stereoselectivity. In every case the more effective d sense orientation directs the -X-Y- grouping toward h-ar, leaving the remainder of the carbon chain in the same conformation and orientation as that of l-II-A during its enzymic hydrolysis. In d(-)-I-A, d(-)- I-B, S(-)-I-C, and d(-)-I-D, the groupings -X-Y- fit well into ar-h and very high reactivity and high stereoselectivity result. The α-heteroatoms of I-A and I-B and I-D, and the planar π-electron structure of I-C permit fit in ar-h, as do the α-heteroatom substituents in V-A and V-B. In I-F and I-G, the α-CH2, like the α-CH3 in V-C, does not permit the -X-Y- grouping to fit easily into h-ar, and low reactivity and low stereoselectivity result, still with d preference. During hydrolysis of these cyclized substrates in the d sense, the am site is unoccupied. When the second ring in a cyclized substrate is eight-membered, hydrolysis in the l sense may be preferred.