Active Site of alpha-Lytic Protease. Enzyme-Substrate Interactions

Abstract

The active site of α-lytic protease has studied with a number of synthetic peptides and compared with similar data published for elastase. The kinetic data indicate that the active site of α-lytic protease extends over at least six subsites (S₄-S′₂. This extended active site has the effect of increasing k_cat/K_m by more than 10⁶-fold on going from an N-acetylated amino acid amide to a hexapeptide, due mainly to increases in k_cat. There are major differences between α-lytic protease and elastase, both in terms of the kinetic parameters for a number of substrates and in terms of the tertiary structure of the active site. The ability of the S ₁ subsite to interact with various P₁ amino acid side chains differs markedly between the two enzymes, and can be rationalized in terms of the tertiary structural differences. For α-lytic protease, enzyme-substrate interactions made in subsite S₂ appears to be of primary importance, whereas subsite S₄ is most important for elastase. The tertiary structural homology of α-lytic protease with another bacterial serine protease, Streptomyces griseus protease A, has allowed detailed model building of a tetrapeptide Ac-Pro-Ala-Pro-Ala-OH at the active site. In this way, the subsites S₁-S₄ have been examined for α-lytic protease and compared to other serine proteases.