Sequential conformation changes of chinese hamster ovary dihydrofolate reductase at its active sites.
The local fluorescence probes, 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) and NADPH were employed to detect urea-induced conformation changes at each active site of dihydrofolate reductase (DHFR), respectively. The results indicate that local conformation change at DHF/TNS could be superimposed by the conformation change calculated from the enzyme activity change with a three-state model; while at NADPH site it is lagged in the first transition. This difference is further supported by the different relative changes of Michaelis constants at 0, 1 and 1.8 M urea for each substrate. Our results suggest that local conformation at DHF site is more flexible than that at NADPH site, and the urea-induced unfolding could be ascribed to a four-state transition.
Zhang
Wen-he
WH
Institute of Biophysics, Academy Sinica, 15 Datun Road, Beijing 100101, China.
Qin
San-bo
SB
Zhang
Hong-jie
HJ
Zhu
Zhi-yong
ZY
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry; D008962:Models, Theoretical; D009249:NADP / Q000737:chemistry; D009282:Naphthalenesulfonates / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D009249:NADP; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase