The main
protease of the coronavirus causing
severe acute respiratory syndrome performs proteolytic processing of the viral
polyproteins. The active form of the
enzyme is a homodimer with each subunit consisting of three structural domains. Domains I and II, hosting the complete catalytic machinery, constitute the N-terminal
chymotrypsin-like folding scaffold and connect to the extra C-terminal domain III by a long loop. Previously, the domain III-truncated
enzyme was demonstrated to fold independently into an intact
chymotrypsin-like fold, but it showed no
enzyme activity. To further delineate the structure-function relationships of the domain III and the long loop, we generated some truncated and mutated M(pro) forms bearing various combinations of the loop with other structural parts of the
enzyme. Their conformational and association properties were investigated in detail. Far-ultraviolet circular dichroism (CD) measurements revealed that these fragments could fold independently. The secondary, tertiary and quaternary structures of these mixtures were monitored by CD, fluorescence spectroscopy and analytical ultracentrifugation. However, no
enzyme activity was observed for any mutant or mixtures. These observations indicate that the covalent linkage between the
chymotrypsin like and the extra domain is essential for enzymatic activity of the main coronavirus
protease and for the integrity of its quaternary structure.