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Probing the Mechansim of Pro Region-Catalyzed Folding using Genetics & Biochemistry
The alpha-lytic protease of the Gram-negative soil bacterium Lysobacter enzymogenes is a serine protease of the chymotrypsin superfamily. The protease is synthesized together with a pro region of similar size that is required for the folding of the protease into its native conformation. The pro region acts as a folding enzyme, catalyzing the transformation of the protein from a molten globule-like ensemble of partially collapsed states into the native protease by lowering the free energy of the transition state of this folding reaction by some 18 kcal/mol. In order to probe the mechanism of this reaction and gain insight into the reason for this unusual folding requirement, I have carried out a genetic screen to isolate mutant versions of alpha-lytic protease whose dependence on the pro region for folding is diminished. I isolated a suppressor of a catalytically defective pro region; the suppressor mutant contains two mutations in residues near each other in the hydrophobic core of alpha-lytic protease. Both mutations are required for the suppression.
The kcat for folding of this mutant protease with the catalytically defective Pro region is increased 1000 fold above that for the folding of the wild-type protease with this Pro region and in fact exceeds that for the folding of the wild-type protease with the wild-type Pro region. I have found in addition that the mutant protease is able to fold 400 times more rapidly than the wild-type protease in the absence of any pro region whatsoever. This acceleration corresponds to a 3.2 kcal/mol reduction in the free energy of the folding transition state. Hence a mutant with improved catalyzed folding shows a substantial improvement in uncatalyzed folding as well. This finding indicates that the transition states for the catalyzed and uncatalyzed folding reactions share certain critical features. |
