Protein structures reflect two tendencies that seem opposed. On one hand, proteins
fold to minimize their free energy. On the other hand, they organize themselves to recognize a ligand.  Minimizing free energy demands well-packed hydrophobic interiors, hydrophilic exteriors, and the
complementation of charged and
polar residues. Maximizing function demands active site clefts where polar groups are sequestered from water, like-charged residues are positioned
close to each other in space, residues adopt strained conformations, and hydrophobic patches are exposed to solvent. This apparent tradeoff between
stability and activity may be stated most strongly as, protein residues that contribute to catalysis or ligand binding are not optimal for protein
stability.
In order to test this hypothesis, we have substituted key active site residues in AmpC β-lactamase, an enzyme that is
well-characterized structurally and mechanistically and measured the changes in stability and activity. Substitutions of key active site residues
Ser64, Lys67, Tyr150, Asn152, and Lys315 decreased the activity of the enzyme by 103-105-fold compared to wild-type, while many
significantly increased the stability of the enzyme, by up to 4.7 kcal/mol.
To determine the structural origins of stabilization, crystal structures of several of these mutants were determined. The crystal
structures AmpC S64D, S64G, K67Q, and N152H were determined to between 1.90 and 1.50 Å resolution. These structures revealed several mechanisms by
which stability was increased, including mimicry of the substrate by the substituted residue (S64D), relief of steric strain (S64G), relief of
electrostatic strain (K67Q), and improved complementarity (N152H).
Our results support the argument that protein residues that contribute to catalysis or ligand binding are not optimal for stability.
Indeed, the preorganization of functionality characteristic of active sites seems to have come at a considerable cost to enzyme stability.
Recent publications: - Wang X, Minasov G, Shoichet BK. Non-covalent interaction energies in covalent complexes: TEM-1 ß-lactamase and ß-lactams. Proteins 47 (1), 86-96 (2002). [Pubmed | DOI | PDB 1JVJ | Download PDF]
- Beadle BM, McGovern SL, Patera A, Shoichet BK. Functional analyses of AmpC ß-lactamase through differential stability. Protein Sci 8 (9), 1816-24 (1999). [Pubmed]
- Shoichet BK, Baase WA, Kuroki R, Matthews BW. A relationship between protein stability and protein function. Proc Natl Acad Sci U S A 92 (2), 452-6 (1995). [Pubmed | PDF | PDB 253L | PDB 254L | PDB 255L]
|
