title: Replacement of Phe274 with conserved residue Tyr274 for reactive center loop expulsion in antithrombin.
abstract: BACKGROUND
  The reactive center loop (RCL) of native antithrombin is partially inserted in the main serpin body. It must be fully exposed for optimal inhibitory function.
  
  
  OBJECTIVE
  To test the hypothesis that P(14)-s2B interaction affects loop insertion in antithrombin. By mutating Phe(274) to Tyr(274), the objective was to introduce P(14)-s2B interaction in antithrombin.
  
  
  METHODS
  Site-directed mutagenesis and affinity chromatography were used to obtain purified recombinant protein. Antithrombin's ability to form sodium dodecyl sulfate (SDS)-stable complex with thrombin, stoichiometry of thrombin inhibition, second-order rate constant for thrombin and factor Xa (fXa) inhibition (M(-1) s(-1)), and heparin dissociation constant (K(D); tryptophan fluorescence emission spectra) were determined.
  
  
  CONCLUSIONS
  A marginal, but inconclusive, difference between the wild type and the mutant was observed. The result highlights the variable effect of P(14)-s2B interaction in different serpins. Alternate hypothesis for achieving loop expulsion is proposed.
authors.affiliation: Center for Molecular Biology of Oral Diseases, College of Dentistry, Chicago, IL, USA. uduhan@uicalumni.org
authors.forename: Urmila
authors.initials: U
authors.lastname: Duhan
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D058833:Antithrombin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D058833:Antithrombin Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine