The reversible oxidation of vicinal SH groups in 4-aminobutyrate aminotransferase. Probes of conformational changes.
4-Aminobutyrate aminotransferase is inactivated by preincubation with iodosobenzoate at pH 7. The reaction of 2 SH residues/dimer resulted in formation of an oligomeric species of Mr = 100,000 detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunits cross-linked via a disulfide bond are dissociated by addition of 2-mercaptoethanol which also restores full catalytic activity (Choi, S. Y., and Churchich, J.E. (1985) J. Biol. Chem. 260, 993-997). The substrate 2-oxoglutarate prevents inactivation of the enzyme by iodosobenzoate and the subsequent formation of one disulfide bond, whereas 4-aminobutyrate has no effect on the reactivity of SH groups with iodosobenzoate. Modified 4-aminobutyrate aminotransferase (containing 1 disulfide bond) catalyzes a half-transamination reaction; but it is unable to react with 2-oxoglutarate to generate the aldimine form of the enzyme. The spectroscopic properties (fluorescence yield and polarization of fluorescence) of PMP bound to the modified enzyme are different from those of pyridoxamine phosphate (PMP) bound to the native enzyme. The polarization of fluorescence values of PMP bound to the cross-linked enzyme, excited over the spectral range 310-370 nm, are greater (25%) than those of the cofactor of the native enzyme. An increase in the polarization values implies that the motion of PMP is restricted when the subunits are cross-linked via a disulfide bond.
Kim
D S
DS
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Churchich
J E
JE
D000612:4-Aminobutyrate Transaminase / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D007463:Iodobenzoates / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine
D000596:Amino Acids; D001051:Apoenzymes; D007463:Iodobenzoates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C008154:2-iodosobenzoic acid; D000612:4-Aminobutyrate Transaminase