Catalysis of a protein folding reaction: thermodynamic and kinetic analysis of subtilisin BPN' interactions with its propeptide fragment.
The in vivo folding of subtilisin is dependent on a 77 amino acid propeptide, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme (Ikemura et al., 1987). We have cloned, expressed, and purified large quantities of the 77 amino acid subtilisin propeptide. This has enabled us to characterize its participation in the subtilisin folding reaction by spectroscopic and microcalorimetric methods. Unfolded subtilisin, when returned to native conditions, is kinetically isolated from its native state. Folding of subtilisin with the native calcium site-A is extremely slow even in the presence of a high concentration of isolated propeptide. The folding of a calcium-free mutant subtilisin, however, is readily catalyzed by the isolated propeptide. The propeptide-subtilisin folding reaction can be described as the following equilibrium: P(u) + S(u)<==>P-S<==>Pf-Sf<==>P(u) + Sf, where S(u) and P(u) are subtilisin and propeptide, respectively, which are largely unstructured at the start of the reaction; P-S is a collision complex of unfolded subtilisin and propeptide; Pf-Sf is the complex of folded subtilisin and propeptide; and Sf is folded subtilisin. The rate-limiting step in the folding reaction of calcium-free mutant subtilisin is formation of the initial collision complex, P-S. The rate at which P(u) and S(u) form a productive collision complex is approximately 500 M-1 s-1. The collision complex appears to be an early folding unit which, once formed, results in rapid isomerization to the fully folded complex. The rate constant for isomerization of the collision complex to the folded complex is > or = 0.5 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Strausberg
S
S
Center for Advanced Research in Biotechnology, Maryland Biotechnology Institute, Rockville 20850.
Alexander
P
P
Wang
L
L
Schwarz
F
F
Bryan
P
P
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D002151:Calorimetry; D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D010446:Peptide Fragments; D011498:Protein Precursors; D013381:Subtilisins