Abstract
The kinetic specificities of BPN' and Carlsberg subtilisins [EC 3. 4. 21. 14] were examined with various nucleus-substituted derivatives of Nα-acetylated aromatic amino acid methyl esters for mapping their hydrophobic binding sites in comparison with that of α-chymotrypsin. The Carlsberg enzyme was generally much more reactive than the BPN' enzyme due to the larger kcat value. The fact that the two subtilisins hydrolyzed Ac-Tyr(PABz)-OMe, which is a derivative of tyrosine bearing a planar traps-p-phenylazobenzoyl group at the OH-function, with the smallest Km value showed that these enzymes possess a more extended aromatic binding site than has so far been demonstrated. Ac-Phe(4-NO2, )-OMe was remarkable in being hydrolyzed with a particularly large kcat value (5, 500±700s-1 at pH 7.8 for Carlsberg subtilisin). Ac-Phe(4-NO2)-OMe and Ac-Tyr-OMe were distinguished by Carlsberg subtilisin in terms of kcat but not by BPN' subtilisin, suggesting that the specificity site of the former is more sensitive to a small change in size of substituent than that of the latter. Ac-Trp(NCps)-OMe and Ac-Trp(NCps)-OH were bound to the enzyme's active site but in a competitive manner. A difference in the standard free energies of binding between the two enzymes may indicate that the hydrophobic cleft of Carlsberg subtilisin is somewhat deeper and/or narrower than that of BPN' subtilisin.
