Kinetic specificities of BPN' and Carlsberg subtilisins. Mapping the aromatic binding site

J Biochem. 1978 Sep;84(3):531-8. doi: 10.1093/oxfordjournals.jbchem.a132157.


The kinetic specificities of BPN' and Carlsberg subtilisins [EC] were examined with various nucleus-substituted derivatives of Nalpha-acetylated aromatic amino acid methyl esters for mapping their hydrophobic binding sites in comparison with that of alpha-chymotrypsin. The Carlsberg enzyme was generally much more reactive than the BPN' enzyme due to the larger kcat value. The fact that the two sutilisins hydrolyzed Ac-Tyr(PABz)-OMe, which is a derivative of tyrosine bearing a planar trans-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 +/- 700 s-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.

Publication types

  • Comparative Study

MeSH terms

  • Bacillus / enzymology
  • Bacillus subtilis / enzymology
  • Binding Sites
  • Chymotrypsin / metabolism
  • Hydrolysis
  • Kinetics
  • Species Specificity
  • Substrate Specificity
  • Subtilisins / antagonists & inhibitors
  • Subtilisins / metabolism*


  • Subtilisins
  • Chymotrypsin