Renaturation of formiminotransferase-cyclodeaminase from guanidine hydrochloride. Quaternary structure requirements for the activities and polyglutamate specificity.
Formiminotransferase-cyclodeaminase denatured in 6 M guanidine hydrochloride (Gdn.HCl) refolds and reassembles to the native octameric structure upon dilution into buffer. Both enzymic activities are recovered to greater than 90%, and the renatured enzyme "channels" the formiminotetrahydropteroylpentaglutamate intermediate. Under conditions where the two activities are recovered simultaneously, the rate-limiting step in reactivation is first order with respect to protein, with k = 1.9 X 10(-5) s-1 at 22 degrees C and delta E approximately equal to 15 kcal mol-1. In the presence of 1.5 M urea, renaturation is arrested at the level of dimers having only transferase activity. Subsequent dialysis to remove the urea leads to recovery of deaminase activity and formation of octamer. Kinetic studies with mono- and pentaglutamate derivatives of the folate substrates demonstrated that native and renatured enzyme as well as deaminase-active dimers [Findlay, W. A., & MacKenzie, R. E (1987) Biochemistry 26, 1948-1954] have much higher affinity for polyglutamate substrates, while the transferase-active dimers do not. These results indicate that the transferase activity is associated with one type of subunit-subunit interaction in the native tetramer of dimers and that the polyglutamate binding site and the deaminase activity are associated with the other interface. A dimeric transferase-active fragment generated by limited proteolysis of the native enzyme can also be renatured from 6 M Gdn.HCl, confirming that it is an independently folding domain capable of reforming one type of subunit interaction.
Findlay
W A
WA
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
MacKenzie
R E
RE
D000642:Ammonia-Lyases / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D011489:Protein Denaturation; D013379:Substrate Specificity
D006146:Guanidines; D046911:Macromolecular Substances; D000642:Ammonia-Lyases; C020148:formiminotetrahydrofolate cyclodeaminase; D019791:Guanidine