Prodomain mutations at the subtilisin interface: correlation of binding energy and the rate of catalyzed folding.

The in vivo folding of subtilisin is dependent on a 77 amino acid prosequence, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme. The recent determination of the structure of a complex of the prodomain and a calcium-free subtilisin mutant has suggested how the prodomain may catalyze subtilisin folding [Bryan, P., Wang, L., Hoskins, J., Ruvinov, S., Strausberg, S., Alexander, P., Almog, O., Gilliland, G., & Gallagher, T. (1995) Biochemistry 34, 10310-10318]. In the complex, the prodomain packs against the two parallel surface helices of subtilisin (residues 104-116 and residues 133-144) and supplies caps to the N-termini of the two helices. The binding site is contained almost entirely in the linear sequence 100-144 of subtilisin. The C-terminus of the prodomain (residues 72-77) extends out from its central part to bind like a substrate in subtilisin's active site cleft. The simplest model of catalyzed folding is one in which the observed binding interaction in the complex accelerates folding by stabilizing an intermediate which includes the 45 amino acid alpha beta alpha substructure in subtilisin. According to our hypothesis, amino acids 100-144 would have a native-like fold in the intermediate which the prodomain stabilizes. Guided by the structure of the bimolecular complex of subtilisin and its prodomain, we have constructed mutations in the C-terminal region of the prodomain. Analysis of five mutants reveals a general correlation between the ability of the prodomain to bind to native subtilisin and its ability to accelerate subtilisin folding.(ABSTRACT TRUNCATED AT 250 WORDS)