Conformational differences between high clotting human alpha-thrombin and nonclotting gamma-thrombin.
The conformations of human alpha-thrombin and gamma-thrombin have been compared by circular dichroism, solvent perturbation different spectroscopy, and chemical modification. Circular dichroism studies indicate that proteolytic conversion of alpha-thrombin to gamma-thrombin is accompanied by considerable conformational changes which include a decrease in alpha-helical content from 5-7% to 0-1%. Solvent perturbation at pH 6.0 obtained with 20% ethylene glycol, 20% glycerol, and 20% dimethyl sulfoxide indicates an apparent exposure of 3.5 +2- 0.2 tryptophan and 7.8 +/- 0.1 tyrosine residues in alpha-thrombin and 4.6 +/- 0.2 tryptophan and 9.2 +/0 0.3 tyrosine residues in gamma-thrombin. This increased exposure is substantiated by the greater reactivity of tryptophan residues in gamma-thrombin toward dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide. It suggests that gamma-thrombin is a less compact molecule than the parent alpha-thrombin. Solvent perturbation studies of alpha-thrombin and gamma=thrombin inhibited by phenyl-methanesulfonyl fluoride showed that 0.3 +/- tryptophan and 0.9 +/- 0.3 tyrosine residues in alpha-thrombin and 0.6 +/- 0.3 tryptophan and 1.3 +/- 0.4 tyrosine residues in gamma-thrombin were blocked by the inhibitor. These subtle differences in the extent of blocking of tyrosine and tryptophan suggest a tighter conformation in the catalytic site of gamma-thrombin compared to that of alpha-thrombin.
D002942:Circular Dichroism; D006801:Humans; D007202:Indicators and Reagents; D010664:Phenylmethylsulfonyl Fluoride; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet; D013452:Sulfonium Compounds; D013917:Thrombin / Q000502:physiology*; D014364:Tryptophan; D014443:Tyrosine; D014508:Urea
D007202:Indicators and Reagents; D013452:Sulfonium Compounds; C024586:dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium; D014443:Tyrosine; D010664:Phenylmethylsulfonyl Fluoride; D014364:Tryptophan; D014508:Urea; D013917:Thrombin