Table I.

Amino acid residues affected and primers used to make mutants of ETA

TypeAmino AcidMutant(s)Mutant Primer(s) Used
Active site mutantsHis72H72A5′-TCTAACAAATAGAGCTATCGCTAAATTTGC-3′
Asp120D120A5′-TGGTGCAGGTGTTGCTTTAGCATTAATCAG-3′
Ser195S195C5′-AGTTCCGGGAAATTGTGGATCAGGTATATT-3′
Active site structure maintenance mutantsPro192P192A5′-TGGATTTACAGTTGCGGGAAATTCTGGATC-3′
Ser211S211A5′-AGTTGGTATACATGCTAGCAAAGTGTCTCA-3′
D-loop mutantsPro162P162A5′-ATTAATAGGCTATGCATTCGATCATAAAGT-3′
Phe163F163G5′-AATAGGCTATCCAGGCGATCATAAAGTTAA-3′
Asp164D164A5′-AGGCTATCCATTCGCTCATAAAGTTAACCA-3′
D164G5′-GGCTATCCATTCGGTCATAAAGTTAAC-3′
Substrate specificity mutantsLys213K213Xa5′-TATACATTCTAGCRVNGTGTCTCATCTTGA-3′
Other mutantsGlu12E12A5′-AAAAAACATGAAGCGAAATGGAATAAG-3′
Ile62I62VGenerated by random mutagenesis
Arg87R87GGenerated by random mutagenesis
  • a Where “X” refers to any amino acid change, and in nucleotide changes, where R = A or G, V = A or C or G, and N = A or C or G or T. Underlined bases are areas in the primer that have been mutated to elicit the corresponding amino acid mutation. A complementary primer for each primer listed here was also made to perform the mutagenesis method outlined in Materials and Methods.