Rationale for engineering an enzyme by introducing a mutation that compensates for a deletion.
Procedures are needed for the manipulation of enzymes to introduce improved catalytic efficiencies and substrate affinities, even in the absence of information on three-dimensional structure. We previously described a selection for amino acid replacements that compensate for a large polypeptide deletion in an enzyme. The rationale is that, because of the missing polypeptide sequence, compensatory replacements must create new enzyme-substrate contacts that are not present in the native protein. Here we introduce into an undeleted enzyme a mutation that compensates for a large deletion in the same enzyme. Binding and kinetic measurements show that this mutation has the same effect on the undeleted as on the deleted protein. The results suggest that a new enzyme-substrate interaction has been created by the mutation and that its contribution to the interaction energy is similar in both the deleted and undeleted molecules. Introduction of this class of mutations into enzymes may in general be useful for engineering them to have improved interactions with ligands and substrates.
Regan
L
L
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Buxbaum
L
L
Hill
K
K
Schimmel
P
P
D000412:Alanine-tRNA Ligase / Q000235:genetics*; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D002872:Chromosome Deletion; D005818:Genetic Engineering; D007700:Kinetics; D008722:Methods; D009154:Mutation; D011487:Protein Conformation; D012344:RNA, Transfer, Ala / Q000378:metabolism; D013329:Structure-Activity Relationship
D012344:RNA, Transfer, Ala; D000604:Amino Acyl-tRNA Synthetases; D000412:Alanine-tRNA Ligase