Sulphydryl group modification of aspartate aminotransferase with 3-bromo-1,1,1-trifluoropropanone during catalysis.
After protection of cysteine-45 and -82 with iodoacetamide or N-ethylmaleimide, and in the presence of saturating concentrations of substrates, the supernatant isozyme of pig heart aspartate transaminase has been covalently modified at cysteine-390 with 3-bromo-1,1,1-trifluoropropanone. The modified enzyme retains 60-70% of the initial specific activity and is similar to native enzyme in pH and temperature stability. After tagging cysteine-390 with the fluorinated compound, the enzyme retains  substrate and inhibitor binding abilities; as shown by direct spectrophotometric titration of the active-site chromophores. The 19F NMR spectrum of the modified enzyme has been obtained by a Fourier transform NMR method. Although the transaminase is a dimeric enzyme, 19F bound at each subunit's cysteine-390 gives rise to only a single 19F resonance upfield from that of trifluoroacetic acid. The fact that the chemical shifts of the 19F probe differ in native and guanidine hydrochloride (Gdn-HCl) denatured enzyme is interpreted as the effect of the native protein groups on the probe. The discordance between the changes induced by varying concentrations of Gdn-HCl on the 19F resonance parameters, on the one hand, and the changes in enzyme activity and prosthetic group absorbance, on the other, suggests that, in aspartate transaminase, cysteine-390 lies in an environment dissimilar from that of the active-site components.
D000096:Acetone / Q000031:analogs & derivatives* / Q000494:pharmacology; D001219:Aspartate Aminotransferases / Q000378:metabolism; D001665:Binding Sites; D001894:Borohydrides; D003488:Cyanogen Bromide; D005033:Ethylmaleimide / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D013438:Sulfhydryl Compounds
D001894:Borohydrides; D006146:Guanidines; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D000096:Acetone; C004269:bromotrifluoroacetone; D001219:Aspartate Aminotransferases; D005033:Ethylmaleimide; D003488:Cyanogen Bromide