Fluorescence studies on the dissociation and denaturation of pigeon liver malic enzyme.
Exposure of pigeon liver malic enzyme [S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) in medium concentrations of guanidine-HCl at 25 degrees C and pH 7.45 caused biphasic conformational changes of the enzyme molecule. Molecular weight determination confirmed that the enzyme tetramers were dissociated to monomers in phase I transition. Enzymatic activity was completely lost in this phase. Recovery of the enzyme activity was only possible in the early stages of the phase I transition. Phase II was due to enzyme unfolding, as judged by circular dichroism and the fluorescence parameters of the enzyme. The steps of the transformation of native malic enzyme into a completely denatured state were in the following sequence: tetramer----monomer----random coil. Extensive denaturation of the enzyme molecule resulted in irreversible aggregation. Dissociation and denaturation were accompanied by a red-shift of the fluorescence spectrum (328----368 nm). Fluorescence quenching studies indicated that tryptophan residues of the enzyme molecule were buried deeply in the interior of the molecule. The tryptophan residues were only partially accessible by acrylamide and almost inaccessible by KI. Dissociation and denaturation were accompanied by exposure of the tryptophan residues, as manifested by the accessibility of the enzyme molecule toward KI or acrylamide.
Lee
H J
HJ
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, China.
Chen
Y H
YH
Chang
G G
GG
D020106:Acrylamide; D000178:Acrylamides; D000465:Algorithms; D000818:Animals; D010856:Columbidae; D005453:Fluorescence; D019791:Guanidine; D006146:Guanidines; D008099:Liver / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008970:Molecular Weight; D011489:Protein Denaturation; D014364:Tryptophan
D000178:Acrylamides; D006146:Guanidines; D020106:Acrylamide; D014364:Tryptophan; D008291:Malate Dehydrogenase; D019791:Guanidine