Replacement of Phe274 with conserved residue Tyr274 for reactive center loop expulsion in antithrombin.
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.
Duhan
Urmila
U
Center for Molecular Biology of Oral Diseases, College of Dentistry, Chicago, IL, USA. uduhan@uicalumni.org
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
D058833:Antithrombin Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine