Parameters affecting the restoration of activity to inactive mutants of thymidylate synthase via subunit exchange: further evidence that thymidylate synthase is a half-of-the-sites activity enzyme.
In a previous study we demonstrated that Escherichia coli thymidylate synthase activity could be restored completely by incubating basically inactive mutants of this enzyme at room temperature with R(126)E, another inactive mutant [Maley, F., Pedersen-Lane, J., and Changchien, L.-M. (1995) Biochemistry 34, 1469-1474]. Since only one of the enzyme's two subunits possessed a functional active site and the restoration of activity could be titrated to be equivalent to that of the wild-type enzyme's specific activity, it was proposed that thymidylate synthase was a half-of-the-sites activity enzyme. We now provide additional support for this thesis by presenting an in-depth analysis of some conditions affecting the restoration of enzyme activity. For this purpose, we employed two mutants with marginal thymidylate synthase activity, Y(94)A and R(126)E. The parameters that were examined included pH, concentration of protein, temperature, and urea concentration, all of which influenced the rate of activity restoration. It was found, surprisingly, that by maintaining the amount of each protein constant, while increasing the volume of solution, the rate and total activity restored was greatly enhanced. Increasing the pH from 6.0 to 9.0 markedly increased the rate at which the optimal activity was restored, as did increasing the temperature from 4 to 40 degrees C. A similar effect was obtained when the incubation of the mutants was conducted at 4 degrees C in the presence of 1.5 M urea, a temperature at which activity is restored extremely slowly. Raising the pH to 9.0 resulted in an almost instantaneous restoration of activity at 4 degrees C. The manner in which thymidylate synthase activity is restored from the mutants in the presence of varying concentrations of ethanol, ethylene glycol, and glycerol suggests that changes in subunit interaction and enzyme conformation are in part responsible for the observed differences. Most significantly, at solution levels of 10%, ethanol was found to activate, while ethylene glycol inhibited slightly and glycerol was somewhat more inhibitory. At a concentration of 20%, ethanol inhibited rather strikingly, ethylene glycol was slightly more inhibitory than at 10%, and glycerol was strongly inhibitory. Since the net result of these findings is the suggestion that the restoration of thymidylate synthase activity is due to a separation of the mutant dimers into their respective subunits, followed by their recombination to an active heterodimer, evidence for this phenomenon was sought by separating the recombined dimers using nondenaturating polyacrylamide gel electrophoresis. Sequence analysis of the isolated homo- and heterodimers clearly demonstrated that the active enzyme is a product of subunit exchange, one that is very efficient relative to the wild-type enzyme, which did not exchange subunits unless denatured.
Saxl
R L
RL
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, New York 12201-0509, USA.
Changchien
L M
LM
Hardy
L W
LW
Maley
F
F
D000409:Alanine / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D000431:Ethanol / Q000737:chemistry; D019855:Ethylene Glycol / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012996:Solutions / Q000737:chemistry; D013696:Temperature; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics; D014508:Urea / Q000737:chemistry
D010446:Peptide Fragments; D012996:Solutions; D000431:Ethanol; D018698:Glutamic Acid; D014443:Tyrosine; D014508:Urea; D001120:Arginine; D013940:Thymidylate Synthase; D019855:Ethylene Glycol; D000409:Alanine; D005990:Glycerol