Acid-induced folding of yeast alcohol dehydrogenase under low pH conditions.
Under conditions of low pH, the conformational states of holo-YADH and apo-YADH were examined by protein intrinsic fluorescence, ANS fluorescence, and far-UV CD measurements. The results obtained show that a low ionic strength, with the addition of HCl, the holo- and apo- YADH denatured gradually to reach the ultimate unfolded conformation in the vicinity of pH 2.0 and 2.5, respectively. With the decrease of pH from 7.0 to 2.0, the fluorescence emission decreased markedly, with its emission maximum red-shifting from 335 to 355 nm, indicating complete exposure of the buried tryptophan residues to the solvent. The far-UV CD spectra show the loss of the arrayed secondary structure, though the acid-denatured enzyme still maintained a partially arrayed secondary structure. A further decrease in pH by increasing the concentration of HClO4 induced a cooperative folding of the denatured enzyme to a compact conformation with the properties of a molten globule, described previously by Goto et al. [Proc. Natl. Acad. Sci. USA 87, 573-577 (1990)]. More extensive studies showed that although apo-YADH and holo-YADH exhibited similar behavior, the folding cooperative ability of apo-YADH was lower than that of the holo-enzyme. From the above results, it is suggested that the zinc ion plays an important role in the proper folding of YADH and in stabilizing its native conformation.
Le
W P
WP
Department of Biology, Xiamen University, China.
Yan
S X
SX
Zhang
Y X
YX
Zhou
H M
HM
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D001051:Apoenzymes / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D010472:Perchlorates / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D001051:Apoenzymes; D005456:Fluorescent Dyes; D010472:Perchlorates; C027132:1-anilino-8-naphthalenesulfonate; D000426:Alcohol Dehydrogenase