Functional Linkage Between the Active Site of α-Lytic Protease and Distant Regions of Structure: Scanning Alanine Mutagenesis of a Surface Loop Affects Activity and Substrate Specificity

Autor: Mace, James E., Wilk, Barry J., Agard, David A.
Zdroj: JMB Online (Journal of Molecular Biology); August 4,1995, Vol. 251 Issue: 1 p116-134, 19p
Abstrakt: Previous structural and kinetic characterization of mutations within the active site of α-lytic protease have demonstrated that amino acid residues in direct contact with the substrate are major substrate specificity determinants. The experiments described here identify residues 216–226 of α-lytic protease as a region of structure peripheral to the active site that also plays an important role in establishing the substrate specificity of the enzyme. Alanine substitution mutations within this surface loop of 19 amino acid residues significantly perturb the enzyme's specificity profile, despite being as far as 21 Å from the hydroxyl group of Ser195. The kinetic consequences of the mutations are remarkably independent of position within the loop and suggest that active site plasticity is affected more than static structure. Kinetic characterization of double mutants with the Met190→Ala broad-specificity active site mutation reveals varying degrees of non-additivity and indicates that active site plasticity can be influenced through multiple sets of interactions. Although these results clearly demonstrate that tuning of serine protease activity is possible through remodelling of structure surrounding the active site, practical issues such as retaining compatibility with the folding mechanism and stability of the mature enzyme present significant obstacles to general application of the technique.
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