Reactivity of one thiol group in the dimeric protein, 4-aminobutyrate aminotransferase.
The enzyme 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) from pig brain is inactivated by incubation with 5-5'-dithiobis-2-nitrobenzoic acid at pH 7.4. The reaction of one SH group per dimer brings about 95% loss of aminotransferase activity. The reaction with 5-5'-dithiobis-2-nitrobenzoic acid under pseudo-first-order conditions proceeds at a slow rate (Kobs = 0.05 min-1) and the substrate 4-aminobutyrate has no effect on the rate of inactivation. The inactivation of the enzyme cannot be related to dissociation of the cofactor (pyridoxal-P and pyridoxamine-P) from the catalytic site. One thiol group of the enzyme reacts with N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid at pH 7.4. The reaction of approximately one SH group per dimer causes 95% loss of aminotransferase activity. The absorption and fluorescence properties of the enzyme tagged with the chromophore were used to gain information on the microenvironment of the reactive SH group critically connected with catalytic activity. The fluorescent properties, emission maximum (470 nm), fluorescence lifetime (19 ns) and degree of exposure to the collisional quencher acrylamide (Kq = 1.8 . 10(8) M-1 . s-1) indicate that the chromophore is shielded by the protein matrix.
D000612:4-Aminobutyrate Transaminase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D000637:Transaminases / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D000637:Transaminases; D000612:4-Aminobutyrate Transaminase