Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea.
The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.
Wang
G F
GF
Department of Biochemistry and Biophysics, College of Life Sciences, Wuhan University, People's Republic of China.
Cao
Z F
ZF
Zhou
H M
HM
Zhao
Y F
YF
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology*; D007700:Kinetics; D045542:PQQ Cofactor; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D015363:Quinolones / Q000378:metabolism; D011809:Quinones / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology*
D015363:Quinolones; D011809:Quinones; D045542:PQQ Cofactor; D014508:Urea; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D019791:Guanidine; D002118:Calcium