Activation of chicken liver dihydrofolate reductase by urea and guanidine hydrochloride is accompanied by conformational change at the active site.
It has been reported that the activation of dihydrofolate reductase (DHFR) from L1210 mouse leukaemia cells by KCl or thiol modifiers is accompanied by increased digestibility by proteinases [Duffy, Beckman, Peterson, Vitols and Huennekens (1987) J. Biol. Chem. 262, 7028-7033], suggesting a loosening up of the general compact structure of the enzyme. In the present study, the peptide fragments liberated from the chicken liver enzyme by digestion with trypsin in dilute solutions of urea or guanidine hydrochloride (GuHCl) have been separated by FPLC and sequenced. The sequences obtained are unique when compared with the known sequence of DHFR and thus allow the points of proteolytic cleavage identified for the urea- and GuHCl-activated enzyme to be at or near the active site. It was also indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene 6-sulfonate that conformational changes at the active site in dilute GuHCl parallel GuHCl activation. The above results indicate that the activation of DHFR in dilute denaturants is accompanied by a loosening up of its compact structure especially at or near the active site, suggesting that the flexibility at its active site is essential for the full expression of its catalytic activity.
Fan
Y X
YX
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Ju
M
M
Zhou
J M
JM
Tsou
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004789:Enzyme Activation; D005453:Fluorescence; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification; D011487:Protein Conformation; D011489:Protein Denaturation; D012996:Solutions; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology*
D005456:Fluorescent Dyes; D006146:Guanidines; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D012996:Solutions; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase; D014357:Trypsin; D019791:Guanidine