The folding and assembly of the dodecameric type II dehydroquinases

The dodecameric type II dehydroquinases (DHQases) have an unusual quaternar y structure in which four trimeric units are arranged with cubic 23 symmetr y. The unfolding and refolding behaviour of the enzymes from Streptomyces c oelicolor and Mycobacterium tuberculosis have been studied. Gel-permeation studies show that, at low concentrations (0.5 M) of guanidinium chloride (G dmC1), both enzymes dissociate into trimeric units, with little or no chang e in the secondary or tertiary structure and with a 15 % loss (S. coelicolo r) or a 55 % increase (M. tuberculosis) in activity. At higher concentratio ns of GdmC1, both enzymes undergo sharp unfolding transitions over narrow r anges of the denaturant concentration, consistent with co-operative unfoldi ng of the subunits. When the concentration of GdmC1 is lowered by dilution from 6 M to 0.55 M, the enzyme from S. coelicolor refolds in an efficient m anner to form trimeric units, with more than 75 % regain of activity. Using a similar approach the M. tuberculosis enzyme regains less than 35% activi ty. From the time courses of the changes in CD, fluorescence and activity o f the S. coelicolor enzyme, an outline model for the refolding of the enzym e has been proposed. The model involves a rapid refolding event in which ap proximately half the secondary structure is regained. A slower folding proc ess follows within the monomer, resulting in acquisition of the full second ary structure. The major changes in fluorescence occur in a second-order pr ocess which involves the association of two folded monomers. Regain of acti vity is dependent on a further associative event, showing that the minimum active unit must be at least trimeric. Reassembly of the dodecameric S. coe licolor enzyme and essentially complete regain of activity can be accomplis hed if the denatured enzyme is dialysed extensively to remove GdmC1. These results are discussed in terms of the recently solved X-ray structures of t ype II DHQases from these sources.