Role of tryptophan 49 in the heparin cofactor activity of human antithrombin III.
To probe the functional role of tryptophan 49 in human antithrombin III, a mutant antithrombin, W49K, has been expressed in baby hamster kidney cells. The mutation reduces the affinity for heparin pentasaccharide by 1.8 kcal mol-1 but does not alter the heparin enhancement of the rate of factor Xa inhibition. 1H NMR spectra of W49K antithrombin show that the structure of the protein and the mode of heparin binding appear to be unaltered by the mutation, although tryptophan 49 is perturbed by heparin binding. 19F NMR spectra of 6-fluorotryptophan-substituted antithrombin show that tryptophan 49 is in a solvent-exposed environment. The heparin-induced fluorescence enhancement of W49K antithrombin is significantly different from that of wild-type antithrombin. Pentasaccharide induces only a 24% enhancement of antithrombin fluorescence, while high affinity heparin induces an enhancement of 40%. The results indicate that tryptophan 49 is probably a heparin contact residue but can be mutated without altering the remaining heparin-antithrombin interactions or the heparin-induced conformational change and resultant activation toward Factor Xa. Hydrophobic as well as charge interactions are thus probably involved in the specificity of the antithrombin-heparin pentasaccharide interaction. The lower fluorescence enhancements suggest that the heparin-induced 40% fluorescence enhancement used as the hallmark of activating heparin species is not the best indicator of the structural change in antithrombin that results in enhancement of the rate of proteinase inhibition.
Gettins
P
P
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Choay
J
J
Crews
B C
BC
Zettlmeiss
G
G
D000818:Animals; D000990:Antithrombin III / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006493:Heparin / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014364:Tryptophan; D000990:Antithrombin III; D006493:Heparin