Extreme functional sensitivity to conservative amino acid changes on enzyme exteriors

J Mol Biol. 2000 Aug 18;301(3):585-95. doi: 10.1006/jmbi.2000.3997.


Mutagenesis studies and alignments of homologous sequences have demonstrated that protein function typically is compatible with a variety of amino-acid residues at most exterior non-active-site positions. These observations have led to the current view that functional constraints on sequence are minimal at these positions. Here, it is shown that this inference assumes that the set of acceptable residues at each position is independent of the overall sequence context. Two approaches are used to test this assumption. First, highly conservative replacements of exterior residues, none of which would cause significant functional disruption alone, are combined until roughly one in five have been changed. This is found to cause complete loss of function in vivo for two unrelated monomeric enzymes: barnase (a bacterial RNase) and TEM-1 beta-lactamase. Second, a set of hybrid sequences is constructed from the 50 %-identical TEM-1 and Proteus mirabilis beta-lactamases. These hybrids match the TEM-1 sequence except for a region at the C-terminal end, where they are random composites of the two parents. All of these hybrids are biologically inactive. In both experiments, complete loss of activity demonstrates the importance of sequence context in determining whether substitutions are functionally acceptable. Contrary to the prevalent view, then, enzyme function places severe constraints on residue identities at positions showing evolutionary variability, and at exterior non-active-site positions, in particular. Homologues sharing less than about two-thirds sequence identity should probably be viewed as distinct designs with their own sets of optimising features.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry*
  • Bacterial Proteins
  • Binding Sites
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Proteus mirabilis / enzymology
  • Ribonucleases / chemistry*
  • Ribonucleases / physiology*
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship
  • Temperature
  • beta-Lactamases / chemistry*
  • beta-Lactamases / physiology*


  • Amino Acids
  • Bacterial Proteins
  • Ribonucleases
  • Bacillus amyloliquefaciens ribonuclease
  • beta-Lactamases
  • beta-lactamase TEM-1