Dimeric intermediates in the dissociation of lactic dehydrogenase.

Abstract

Rate-determining second-order reactions in the reconstitution of a number of oligomeric enzymes have shown that catalytic activity requires association. In order to answer the question whether in the case of tetrameric enzymes, e.g. lactic dehydrogenase, the bimolecular step belongs to the dimerization of the monomer or the dimer, attempts have been made to isolate intermediates of dissociation, and to study their reconstitution to the native tetramer. Varying concentrations of urea or guanidine · HCl were applied in order to labilize the native quaternary structure; incubation time, temperature and enzyme concentration were changed as additional variables. Both isoenzymes, M4 from pig and bovine skeletal muscle, and H4 from pig heart show separable steps in their denaturation profiles on changing the concentration of urea or guanidine · HCI. At high denaturant concentrations, denatured and deactivated subunits are formed. At about 2 M urea or 0.8–1.2 M guanidine · HCI (t≦ 5° C) metastable intermediates are obtained which represent inactive dimers, as shown by sedimentation analysis. Increased temperature yields wrong aggregates rather than low-molecular-weight intermediates, causing a drastic decrease of reactivation. Reconstitution of the metastable dimeric intermediates to reestablish the native tetramer is characterized by the same rate constants which have been reported previously for the overall reconstitution, starting from denatured monomers [R. Jaenicke (1979) FEBS Symp. 52, 187–198]. In the case of the isoenzyme M4 from pig skeletal muscle (after incubation in 1 M guanidine · HCl) reconstitution follows a simple bimolecular mechanism, while in the case of H4 from pig heart sigmoidal reactivation profiles indicate a more complicated mechanism involving rate-determining first-order processes. The fact there is no significant difference between the kinetics of reactivation during the dimer tetramer transition on the one hand, and the monomer tetramer overall reconstitution on the other, strongly suggests that the rate-determining step in the consecutive association reaction underlying reactivation as well as tetramer formation must be the dimerization of inactive dimers.