L1210 dihydrofolate reductase. Kinetics and mechanism of activation by various agents.
Dihydrofolate reductase from a methotrexate-resistant subline (R6) of L1210 mouse leukemia cells is activated (i.e. has its catalytic activity increased severalfold) by treatment with (a) sulfhydryl-modifying agents (p-chloromercuribenzoate (pCMB) or 5,5'-dithiobis(2-nitrobenzoic acid], (b) salts (KCl or NaCl), or (c) chaotropes (urea or guanidinium hydrochloride). With b or c activation is rapid (less than 10 s), but with a the process is much slower; at 25 degrees C, pseudo first-order rate constants for activation by excess pCMB or 5,5'-dithiobis(2-nitrobenzoic acid) are 0.45 and 0.08 min-1, respectively. Activation can also be monitored by conformational changes in the protein as indicated by enhanced fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate or by increased intrinsic fluorescence of tryptophan residues in the enzyme. Pseudo first-order rate constants for the pCMB-induced conformational change, measured by these fluorimetric procedures (0.45 min-1 and about 0.4 min-1, respectively), are in good agreement with the value obtained from the increase in catalytic activity. The rate of modification of the single cysteine residue in the enzyme by excess 14C-labeled pCMB, however, is faster than the rate of activation, indicating that the conformational change follows derivatization and is the rate-limiting step in the overall process. Activated forms of the enzyme are more labile to thermal denaturation or proteolysis than the untreated enzyme; the former process, however, is retarded by the presence of bovine serum albumin. Activation by the various agents is considered to involve a common mechanism in which interaction of the enzyme with the agents is followed by conformational changes in the enzyme, producing a series of forms that differ in microstructure, catalytic activity, and lability.
Duffy
T H
TH
Beckman
S B
SB
Peterson
S M
SM
Vitols
K S
KS
Huennekens
F M
FM
D000818:Animals; D002460:Cell Line; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology*; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D006146:Guanidines; D012965:Sodium Chloride; D020245:p-Chloromercuribenzoic Acid; D011189:Potassium Chloride; D014508:Urea; D004228:Dithionitrobenzoic Acid; D013762:Tetrahydrofolate Dehydrogenase; D010447:Peptide Hydrolases; D019791:Guanidine