Abstract：
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Critz WJ,Martinez-Carrion Mdoi
10.1021/bi00627a004subject
Has Abstractpub_date
1977-04-19 00:00:00pages
1554-8issue
8eissn
0006-2960issn
1520-4995journal_volume
16pub_type
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