Abstract
In order to investigate how changes in the structures of side-chain aromatic groups of specific substrates influence binding and kinetic specificity in a-chymotrypsin [EC 3.4.21.1]-catalyzed reactions, a number of nucleus-substituted derivatives of the specific ester substrates were prepared and steady-state kinetic studies were carried out at pH 6.5 and 7.8.
Ac-Trp(NCps)-OMe was hydrolyzed more readily at low substrate concentration than Ac-Trp-OMe due to its smaller Km(app) value, suggesting that the bulky 2-nitro-4-carboxyphenylsulfenyl moiety interacts with outer residues rather than with those in the hydrophobic pocket and that this interaction increases the binding specificity. Inhibition experiments using the corresponding carboxylate and analogous inhibitors, however, showed that the carboxy group at the para position of the phenyl nucleus of the substituent sterically hinders association with the active site of α-chymotrypsin at pH 7.8 but not at pH 6.5. The kcat values of Ac-Trp(CHO)-OMe, Ac-Tyr(3-NO2)-OMe, and Ac-m-Tyr-OMe were much higher than those of the corresponding specific substrates, indicating that derivatives with a substituent as large as a formyl, nitro or hydroxyl group at the ε-position are stereochemically favorable to the catalytic process. Remarkable increases in Km(app) were also observed. The individual pa-rameters for Ac-Dopa-OMe, however, were comparable to those for Ac-Tyr-OMe. The hydroxyl function at the pare position thus plays some role in decreasing Km(app) as well as kcat when a substituent of limited size, such as a hydroxyl group, is present at the meta position. Derivatives of Ac-Phe-OMe with a chain of four atoms at the meta or para position of the phenyl nucleus and 2, 3-dihydropyrrolo[2, 3-b]indoles derived from L and D-Ac-Trp-OMe were not hydrolyzed at all. The low reactivity of Ac-o-Tyr-OMe with a large Km(app) value (19 mM at pH 7.8) appears to be as-sociated with the steric effect of the hydroxyl group at the ortho position on intra-molecular rotation around the β-γ bound, in addition to intermolecular interaction. Ac-Trp(OH)-OMe was less reactive than Ac-Trp-OMe but its Km(app) value (1.0 mM) was much smaller than that of Ac-o-Tyr-OMe, which is regarded as geometrically homologous to Ac-Trp(OH)-OMe with respect to the position of modification. The distortion of the indolinone ring arising from the tetrahedral structure at the β-position seems not to bring about a large increase in Kn(app). Unlike the other inhibitors examined, the K; values of Ac-Trp(CHO)-OH and Ac-Tyr(3-NO2)-OH were lower than the Km(app) values of the corresponding substrates. This is also attributed to the steric effect of the substituent at the ε-position.
