Assembly of the catalytic trimers of aspartate transcarbamoylase from unfolded polypeptide chains.
In an effort to stimulate the in vivo formation of active enzyme from newly synthesized polypeptide chains, we have studied the in vitro assembly of the active catalytic subunits of aspartate transcarbamoylase from unfolded polypeptide chains. Hydrodynamic and spectroscopic measurements showed that incubating the catalytic trimers in 4.7 M urea for 45 min at 9 degrees C produced unfolded polypeptide chains largely devoid of the secondary and tertiary structures characteristic of native subunits. Dilution of the urea solutions led to the slow restoration of enzyme activity and the formation of trimers at a rate which could be measured quantitatively by a hybridization technique using succinylated polypeptide chains as a "chase" to "stop" the assembly. Kinetic studies showed that reactivation and assembly of trimers were coincident with a half-time for completion of about 50 min at 0 degrees C. Also, the rate-limiting reaction was first order. Although these results suggest that chain folding is the slow process, spectroscopic studies indicated that large changes in the environments of the aromatic amino acid residues occur very rapidly. Indeed the changes in the absorption spectrum are largely complete before significant reactivation and trimer formation occur. The results are consistent with an assembly mechanism in which the first step is the rapid collapse of the expanded randomly coiled chains to give partially folded monomers. These monomers are not enzymically active and cannot associate to form trimers until a rate-limiting conformational change occurs. Subsequent to this slow process, the "competent" monomers associate via a series of reactions to form active trimers.
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D014508:Urea
D046911:Macromolecular Substances; D012996:Solutions; D014508:Urea; D001221:Aspartate Carbamoyltransferase