Folding Pathway Mediated by an Intramolecular Chaperone (2000)

The subtilisin propeptide functions as an intramolecular chaperone (IMC) that facilitates correct folding of the catalytic domain while acting like a competitive inhibitor of proteolytic activity. Upon completion of folding, subtilisin initiates IMC degradation to complete precursor maturation. Existing data suggest that the chaperone and inhibitory functions of the subtilisin IMC domain are interdependent during folding. Based on x-ray structure of the IMC-subtilisin complex, we introduce a point mutation (E112A) to disrupt three hydrogen bonds that stabilize the interface between the protease and its IMC domain. This mutation within subtilisin does not alter the folding kinetics but dramatically slows down autoprocessing of the IMC domain. Inhibition of E112A-subtilisin activity by the IMC added in trans is 35-fold weaker than wild-type subtilisin. Although the IMC domain displays substantial loss of inhibitory function, its ability to chaperone E112A-subtilisin folding remains intact. Our results show that (i) the chaperone activity of the IMC domain is not obligatorily linked with its ability to bind with and inhibit active subtilisin; (ii) degradation and not autoprocessing of the IMC domain is the rate-limiting step in precursor maturation; and (iii) the Glu112 residue within the IMC-subtilisin interface is not crucial for initiating folding but is important in maintaining the IMC structure capable of binding subtilisin. The subtilisin propeptide functions as an intramolecular chaperone (IMC) that facilitates correct folding of the catalytic domain while acting like a competitive inhibitor of proteolytic activity. Upon completion of folding, subtilisin initiates IMC degradation to complete precursor maturation. Existing data suggest that the chaperone and inhibitory functions of the subtilisin IMC domain are interdependent during folding. Based on x-ray structure of the IMC-subtilisin complex, we introduce a point mutation (E112A) to disrupt three hydrogen bonds that stabilize the interface between the protease and its IMC domain. This mutation within subtilisin does not alter the folding kinetics but dramatically slows down autoprocessing of the IMC domain. Inhibition of E112A-subtilisin activity by the IMC added in trans is 35-fold weaker than wild-type subtilisin. Although the IMC domain displays substantial loss of inhibitory function, its ability to chaperone E112A-subtilisin folding remains intact. Our results show that (i) the chaperone activity of the IMC domain is not obligatorily linked with its ability to bind with and inhibit active subtilisin; (ii) degradation and not autoprocessing of the IMC domain is the rate-limiting step in precursor maturation; and (iii) the Glu112 residue within the IMC-subtilisin interface is not crucial for initiating folding but is important in maintaining the IMC structure capable of binding subtilisin. intramolecular chaperone anilino-8-napthalene sulfonic acid phenylmethylsulfonyl fluoride dithiothreitol Subtilisin E is an alkaline serine protease found inBacillus subtilis (1.Wong S.L. Doi R.H. J. Biol. Chem. 1986; 261: 10176-10181Abstract Full Text PDF PubMed Google Scholar). In vivo this protein exists as a precursor, namely pre-pro-subtilisin (1.Wong S.L. Doi R.H. J. Biol. Chem. 1986; 261: 10176-10181Abstract Full Text PDF PubMed Google Scholar). The presequence acts as a signal peptide and facilitates the secretion of pro-subtilisin across the cytoplasmic membrane, whereas the pro-region or propeptide functions as an intramolecular chaperone (IMC)1 that guides correct folding of the subtilisin domain (2.Ikemura H. Takagi H. Inouye M. J. Biol. Chem. 1987; 262: 7859-7864Abstract Full Text PDF PubMed Google Scholar, 3.Ikemura H. Inouye M. J. Biol. Chem. 1988; 263: 12959-12963Abstract Full Text PDF PubMed Google Scholar, 4.Zhu X. Ohta Y. Jordan F. Inouye M. Nature. 1989; 339: 483-484Crossref PubMed Scopus (322) Google Scholar). Upon completion of folding the precursor removes the IMC domain through autoproteolysis to give active subtilisin (5.Shinde U.P. Inouye M. Trends Biochem. Sci. 1993; 18: 442-446Abstract Full Text PDF PubMed Scopus (163) Google Scholar). Similar folding mechanisms exist in numerous prokaryotic and eukaryotic proteases (6.Barr P. Cell. 1991; 66: 1-3Abstract Full Text PDF PubMed Scopus (555) Google Scholar) that include subtilisin (2.Ikemura H. Takagi H. Inouye M. J. Biol. Chem. 1987; 262: 7859-7864Abstract Full Text PDF PubMed Google Scholar, 3.Ikemura H. Inouye M. J. Biol. Chem. 1988; 263: 12959-12963Abstract Full Text PDF PubMed Google Scholar, 4.Zhu X. Ohta Y. Jordan F. Inouye M. Nature. 1989; 339: 483-484Crossref PubMed Scopus (322) Google Scholar), α-lytic protease (7.Silen J.L. Agard D.A. Nature. 1989; 341 (264): 362Crossref Scopus (197) Google Scholar,8.Baker D. Sohl J.L. Agard D.A. Nature. 1992; 356: 263-265Crossref PubMed Scopus (286) Google Scholar), aqualysin (9.Lee Y.C. Miyata Y. Terada I. Ohta T. Matsuzawa H. Agric. Biol. Chem. 1991; 55: 3027-3032PubMed Google Scholar, 10.Kwon S.T. Terada I. Matsuzawa H. Ohta T. Eur. J. Biochem. 1988; 73: 1-7Google Scholar), carboxypeptidase Y (11.Sørensen P. Winther J.R. Kaarsholm N.C. Biochemistry. 1993; 32: 12160-12166Crossref PubMed Scopus (25) Google Scholar, 12.Winther J.R. Sørensen P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9330-9334Crossref PubMed Scopus (184) Google Scholar), cathepsin L (13.Smith S.M. Gottesman M.M. J. Biol. Chem. 1989; 264: 20487-20495Abstract Full Text PDF PubMed Google Scholar), and thermolysin (14.Marie-Claire C. Ruffet E. Beaumont A. Roques B.P. J. Mol. Biol. 1999; 285: 1911-1915Crossref PubMed Scopus (67) Google Scholar). In addition to proteases, a variety of other proteins such as nerve growth factor (15.Suter U. Angst C. Tien C.L. Drinkwater C.C. Lindsay R.M. Shooter E.M. J. Neurosci. 1992; 12: 306-318Crossref PubMed Google Scholar), amphiregulin (16.Thorne B.A. Plowman G.D. Mol. Cell. Biol. 1994; 14: 1635-1646Crossref PubMed Scopus (58) Google Scholar), and activin A (17.Gray A.M. Mason A.J. Science. 1990; 247: 1328-1330Crossref PubMed Scopus (224) Google Scholar) mature from higher molecular weight precursor proteins. Although propeptides of several proteins can function as IMCs, not all propeptides can directly catalyze folding reactions (18.Eder J. Fersht A.R. Mol. Microbiol. 1995; 16: 609-614Crossref PubMed Scopus (169) Google Scholar, 19.Shinde, U. P., Liu, J. J., and Inouye, M. (1997) Molecular Chaperones in the Life Cycle of Proteins, 1st Ed., pp. 467–490, 2000, 11(1), 3544, Marcel Dekker, Inc., New YorkGoogle Scholar). Such propeptides can exert their biological functions using a myriad of mechanisms (19.Shinde, U. P., Liu, J. J., and Inouye, M. (1997) Molecular Chaperones in the Life Cycle of Proteins, 1st Ed., pp. 467–490, 2000, 11(1), 3544, Marcel Dekker, Inc., New YorkGoogle Scholar). Close scrutiny of such propeptides suggests that they may actually be post-translational “modulators” of protein function because they are directly or indirectly involved in the structural organization (20.Shinde U.P. Inouye M. Semin. Cell Dev. Biol. 2000; 11: 35-44Crossref PubMed Scopus (136) Google Scholar). The conservation of propeptide function across unrelated protease families suggests that, similar to proteases, propeptides may have evolved in multiple parallel pathways and may share a similar mechanism of action (18.Eder J. Fersht A.R. Mol. Microbiol. 1995; 16: 609-614Crossref PubMed Scopus (169) Google Scholar). It is also possible that propeptides have evolutionarily preceded the formation of the protease families (21.Shinde U.P Inouye M. J. Biochem. (Tokyo). 1994; 115: 629-636Crossref PubMed Scopus (50) Google Scholar). IMC-mediated protein folding is best understood in subtilisin and α-lytic protease (20.Shinde U.P. Inouye M. Semin. Cell Dev. Biol. 2000; 11: 35-44Crossref PubMed Scopus (136) Google Scholar). The in vitro folding pathway of pro-subtilisin involves precursor folding (22.Shinde U.P. Li Y. Chatterjee S. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6924-6928Crossref PubMed Scopus (84) Google Scholar, 23.Shinde U.P. Inouye M. J. Mol. Biol. 1995; 252: 25-30Crossref PubMed Scopus (53) Google Scholar), followed by autoprocessing (24.Shinde U.P. Inouye M. J. Mol. Biol. 1995; 247: 390-395Crossref PubMed Scopus (52) Google Scholar) that coincides with structural changes that lead to degradation of the IMC domain (23.Shinde U.P. Inouye M. J. Mol. Biol. 1995; 252: 25-30Crossref PubMed Scopus (53) Google Scholar). Folding of denatured subtilisin in the absence of its IMC domain traps the protease into an inactive molten-globule like intermediate state (8.Baker D. Sohl J.L. Agard D.A. Nature. 1992; 356: 263-265Crossref PubMed Scopus (286) Google Scholar, 23.Shinde U.P. Inouye M. J. Mol. Biol. 1995; 252: 25-30Crossref PubMed Scopus (53) Google Scholar, 25.Eder J. Rheinnecker M. Fersht A. Biochemistry. 1993; 32: 18-26Crossref PubMed Scopus (125) Google Scholar, 26.Eder J. Rheinnecker M. Fersht A. J. Mol. Biol. 1993; 233: 293-304Crossref PubMed Scopus (119) Google Scholar). Addition of the IMC domain in trans helps this intermediate adopt an active conformation (8.Baker D. Sohl J.L. Agard D.A. Nature. 1992; 356: 263-265Crossref PubMed Scopus (286) Google Scholar, 25.Eder J. Rheinnecker M. Fersht A. Biochemistry. 1993; 32: 18-26Crossref PubMed Scopus (125) Google Scholar). Recent data show that the IMC domain can impart structural information onto its protease (27.Shinde U.P. Liu J.J. Inouye M. Nature. 1997; 389, 5: 20-522Google Scholar) and that this imprinting occurs after autoprocessing but before degradation of the IMC from the precursor (28.Shinde U.P. Fu X. Inouye M. J. Biol. Chem. 1999; 274: 15615-15621Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Such results suggest that IMCs promote protein folding by direct stabilization of the rate-limiting folding transition state and support the notion that the IMC is essential only during late stages of the folding process (29.Ellis R.B. Trends Biochem. Sci. 1998; 23: 43-45Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 30.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5194) Google Scholar). Amino acid residues Asp32, His64, and Ser221 constitute the catalytic triad of subtilisin and mediate both autoprocessing and degradation of the IMC domain (3.Ikemura H. Inouye M. J. Biol. Chem. 1988; 263: 12959-12963Abstract Full Text PDF PubMed Google Scholar, 4.Zhu X. Ohta Y. Jordan F. Inouye M. Nature. 1989; 339: 483-484Crossref PubMed Scopus (322) Google Scholar, 5.Shinde U.P. Inouye M. Trends Biochem. Sci. 1993; 18: 442-446Abstract Full Text PDF PubMed Scopus (163) Google Scholar,31.Li Y. Inouye M. J. Biol. Chem. 1994; 269: 4169-4174Abstract Full Text PDF PubMed Google Scholar). However, these two activities are distinctly different from one another. As a result the IMC-S221C-subtilisin can autoprocess its IMC domain but is unable to in the formation of a IMC-subtilisin Y. Inouye M. J. Biol. Chem. 1994; 269: 4169-4174Abstract Full Text PDF PubMed Google Scholar). The x-ray structure of such a T. P. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, U.P. Li Y Inouye M. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). It is from this structure that degradation of the IMC domain to autoproteolysis is essential because the of the IMC domain with the of the protease through numerous hydrogen The the activity of the protease by of its active T. P. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, U.P. Li Y Inouye M. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). However, the of a peptide within the IMC can dramatically its for the protease domain in protease Y. Jordan F. Inouye M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). It is important to that the IMC domain and its are of and from subtilisin (22.Shinde U.P. Li Y. Chatterjee S. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6924-6928Crossref PubMed Scopus (84) Google Scholar, 25.Eder J. Rheinnecker M. Fersht A. Biochemistry. 1993; 32: 18-26Crossref PubMed Scopus (125) Google Scholar, P. U.P. Inouye M. Chaperones and Scholar). between IMC-subtilisin to be important to stabilize the through within the IMC domain suggest the of a direct between the chaperone function and the of proteolytic activity Y. Jordan F. Inouye M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, P. U.P. Inouye M. Chaperones and Scholar). In the we the IMC domain but three hydrogen bonds that exist between the Glu112 and the of the IMC domain. with to the that stabilize the IMC-subtilisin Our results that the does not the folding of This precursor into an active and the does not maturation. However, the autoprocessing and degradation of the IMC domain are dramatically This result is to the of the precursor to a IMC-subtilisin because of the IMC a competitive inhibitor of the subtilisin as the is and these results show that the chaperone and inhibitory functions of the IMC domain are not obligatorily The from The sulfonic acid fluoride and the for from The on an using from of these using by The of the by the to introduce the into (27.Shinde U.P. Liu J.J. Inouye M. Nature. 1997; 389, 5: 20-522Google Scholar) and Y. Inouye M. J. Biol. Chem. 1994; 269: 4169-4174Abstract Full Text PDF PubMed Google Scholar, Y. Jordan F. Inouye M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) using the from The through on an from and into and in with (27.Shinde U.P. Liu J.J. Inouye M. Nature. 1997; 389, 5: 20-522Google Scholar). an of A the proteins by the by and with a and The in of with of a of protease and I. The using a The for and the and the are the of the the of are of As a the protein in (27.Shinde U.P. Liu J.J. Inouye M. Nature. 1997; 389, 5: 20-522Google Scholar, U.P. Fu X. Inouye M. J. Biol. Chem. 1999; 274: 15615-15621Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Y. Inouye M. J. Biol. Chem. 1994; 269: 4169-4174Abstract Full Text PDF PubMed Google Scholar). that the protein of the the with to of The in of by The by for and the to the proteins. The of A with one The protein for to proteins. protein to a that with A. The with A proteins in the and the protein using a in A. pro-subtilisin between and and the and to by an using a The proteins as but this using The protein for and the to a that with the with proteins using a of in the precursor and as and The precursor proteins denatured in The for and folding through of of the protein into of The kinetics of folding by the denatured into a and the changes in with a of for and the of three using the found to a The for the data and proteins are into a and of that the with one This is in a complete of is (23.Shinde U.P. Inouye M. J. Mol. Biol. 1995; 252: 25-30Crossref PubMed Scopus (53) Google Scholar, U.P. Liu J.J. Inouye M. Nature. 1997; 389, 5: 20-522Google Scholar, U.P. Fu X. Inouye M. J. Biol. Chem. 1999; 274: 15615-15621Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Y. Inouye M. J. Biol. Chem. 1994; 269: 4169-4174Abstract Full Text PDF PubMed Google Scholar). The proteins to in the for to Y. Inouye M. J. Biol. Chem. 1994; 269: 4169-4174Abstract Full Text PDF PubMed Google Scholar) and using an with a of the is in of the the between and activity of subtilisin is by the through changes in Y. Jordan F. Inouye M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). are using a to the is as the activity A of to of on an The from to The protein and a of In from to for for Folding kinetics by of precursor proteins into of and changes in the on an and (27.Shinde U.P. Liu J.J. Inouye M. Nature. 1997; 389, 5: 20-522Google Scholar). The proteins and the from to The and the the the proteins and the from to The pathway of subtilisin involves stages (28.Shinde U.P. Fu X. Inouye M. J. Biol. Chem. 1999; 274: 15615-15621Abstract Full Text Full Text PDF PubMed Scopus (72) Google (i) folding of the protease domain that is by its IMC (ii) autoprocessing of the peptide between the of the IMC domain and the of subtilisin; (iii) structural changes within the during IMC to and degradation of the IMC domain that within the catalytic triad of subtilisin the of on this the catalytic residue Ser221 is with the precursor folding, and structural changes to its to give a in the IMC domain remains with subtilisin through Y. Inouye M. J. Biol. Chem. 1994; 269: 4169-4174Abstract Full Text PDF PubMed Google Scholar). A the x-ray structure of such an IMC-S221C-subtilisin U.P. Li Y Inouye M. J. Mol. Biol. 1998; PubMed Scopus Google Scholar), is similar to the by the IMC domain to subtilisin T. P. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the IMC domain the two parallel of the mature domain. are hydrogen bonds that the IMC with mature subtilisin. include the of the IMC domain that with the active of subtilisin. The IMC domain a structure with and to the protease domain T. P. 1995; Full Text Full Text PDF PubMed Scopus Google U.P. Li Y Inouye M. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). Glu112 three hydrogen bonds with the of residues and within the IMC domain and to a in maintaining the IMC domain with subtilisin in a The interface is also by between the interface of the of the IMC domain and the two in subtilisin the of the structure of the complex, that the within subtilisin by the IMC domain P. J. S. T. Biochemistry. 1995; PubMed Scopus Google Scholar). to the of Glu112 in of the two parallel with the in the IMC domain in an and to its in the this residue with an using This protein is to the of Glu112 on the pathway of The in and that wild-type IMC-subtilisin and the IMC-S221C-subtilisin can autoprocess but not its IMC acid within this one to the of on autoprocessing and the of the in the absence of IMC degradation (23.Shinde U.P. Inouye M. J. Mol. Biol. 1995; 252: 25-30Crossref PubMed Scopus (53) Google Scholar). The wild-type and and using a (23.Shinde U.P. Inouye M. J. Mol. Biol. 1995; 252: 25-30Crossref PubMed Scopus (53) Google Scholar, Y. Inouye M. J. Biol. Chem. 1994; 269: 4169-4174Abstract Full Text PDF PubMed Google Scholar). IMC-subtilisin and can to give active mature subtilisin The and of the protease domain in both wild-type and E112A-subtilisin using that the of the two proteins are E112A-subtilisin displays a in the This suggests that the does not dramatically alter the structure of the protease domain. The of the two proteins using and the show similar However, E112A-subtilisin a in the in the of the three two of may be directly by the the of the two proteins The proteins and the of the proteins from to The and and a for such changes can be to in the within residues by the This residue is to and in with The by in of E112A-subtilisin and suggests that changes in can as a result of the However, the and the of both wild-type and E112A-subtilisin are the two proteins also not that can bind with a not It is important to that of for folding, the of mature E112A-subtilisin is than that of the wild-type subtilisin. the of the that to be in of A than that for the wild-type subtilisin The protease than the wild-type protein as using not This may be to of within and the of mature E112A-subtilisin may be to its as with that of wild-type subtilisin. the Glu112 within subtilisin the precursor to into an active protease domain with to that of the wild-type subtilisin with structural of the wild-type and for subtilisin for in a the of the interface on the of the in The proteins and as and IMC-S221C-subtilisin using a that of of the two proteins of and to these of IMC-S221C-subtilisin A that the to IMC-S221C-subtilisin to its IMC domain. However, the IMC domain is not and remains with the protease domain through In of the of is but of autoprocessing of both these in the absence of degradation of the IMC a of after of A However, as mature E112A-subtilisin that the wild-type active the of mature E112A-subtilisin than wild-type subtilisin the active IMC-S221C-subtilisin and are only to autoprocess but not their IMC whereas IMC-subtilisin and can both autoprocess and their IMC the of subtilisin may as a of degradation of the precursor during to the from the precursor for of the protein through results in the of the whereas the IMC domain remains in the not This suggests that the between the IMC domain and to be weaker than in of IMC-S221C-subtilisin the the of autoprocessing and of the and IMC-S221C-subtilisin in their the proteins for their ability to bind to It that can bind with within proteins and to the of the folding of with the a in the from to with a in the that is to the of within the is that is than and this may the process of during the protein It is important to that the binding in the does not as a result of different of is that both and autoprocess of their IMC after is with Y. Inouye M. J. Biol. Chem. 1994; 269: 4169-4174Abstract Full Text PDF PubMed Google Scholar). to but inactive E112A-subtilisin to a with the IMC the It that subtilisin can with the IMC domain to a Jordan F. J. Biol. Chem. Full Text PDF PubMed Scopus Google Scholar). of the IMC added to the wild-type and and the for to The of the proteins using The displays an loss in with the wild-type both mature wild-type and E112A-subtilisin whereas the with the IMC domain is to that the in the of the because of in their IMC displays the between the E112A-subtilisin and its with the IMC domain in the wild-type Although the IMC domain with wild-type subtilisin displays an the IMC domain with E112A-subtilisin displays a different that not the IMC domain in the E112A-subtilisin is that the between Glu112 and the peptide of residues within the IMC is for maintaining the IMC It be that the Glu112 may the of the folding The folding kinetics of the precursor protein and its can be by of denatured IMC-subtilisin in and into of while the changes in using as an for the changes in structure during the process of the are not It is important to that activity of wild-type IMC-subtilisin can be after folding. the kinetics of IMC-subtilisin to be and can be using The of this and the signal of the wild-type precursor protein after of of the signal that complete The folding for both IMC-subtilisin and can be to a using the The folding from the data are and for IMC-subtilisin and displays the structure of the proteins after of IMC-subtilisin and the structure of IMC-subtilisin is and similar to that of the that However, displays a in and a in and is with the by addition of the IMC domain in trans the autoprocessing activity of the protease domain to in the As from wild-type precursor an of its precursor within the of the of the precursor of its IMC domain proteolytic of the does not autoprocessing can and The binding and the active in both as as complex, are not to protease like subtilisin inhibitor or (3.Ikemura H. Inouye M. J. Biol. Chem. 1988; 263: 12959-12963Abstract Full Text PDF PubMed Google Scholar). Such are to be unable to autoprocessing and degradation of the IMC domain (3.Ikemura H. Inouye M. J. Biol. Chem. 1988; 263: 12959-12963Abstract Full Text PDF PubMed Google Scholar). the active of subtilisin in an or precursor its inhibitory IMC domain. proteolytic activity is not during the of the loss in signal because of degradation does not and not the of kinetics of IMC-subtilisin during the of the of a to the of mature subtilisin of its inhibitory IMC domain. the of the mature protease domain as a function of for both IMC-subtilisin and The wild-type precursor can its IMC domain to activity in after these the precursor However, does not during the an the wild-type precursor complete whereas the of Based on we suggest the of the hydrogen bonds between Glu112 within subtilisin and the peptide of the IMC domain does not dramatically alter the folding of the precursor proteins. However, Glu112 autoprocessing of the precursor protein and suggests that the hydrogen between Glu112 and the IMC domain is not important for the folding process but occurs late during the folding pathway to autoprocessing of the This is with data that show the structure of the IMC-subtilisin before and after autoprocessing is different (23.Shinde U.P. Inouye M. J. Mol. Biol. 1995; 252: 25-30Crossref PubMed Scopus (53) Google Scholar, U.P. Inouye M. J. Mol. Biol. 1995; 247: 390-395Crossref PubMed Scopus (52) Google Scholar, U.P. Fu X. Inouye M. J. Biol. Chem. 1999; 274: 15615-15621Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). results also suggest that degradation of the IMC domain to be the rate-limiting step in the the of Glu112 in the binding of the IMC with the protease the ability of the IMC to inhibit protease activity The activity of both the wild-type and E112A-subtilisin are similar of wild-type and E112A-subtilisin using of the IMC domain. A the of as a function of E112A-subtilisin the different of the IMC domain and activity can be within for of the addition of complete degradation of the IMC domain and the of degradation that of the However, in the of wild-type the in is dramatically As from the in wild-type subtilisin of the IMC domain in whereas the of mature E112A-subtilisin the IMC domain in Although the of the IMC domain for E112A-subtilisin not these results show that E112A-subtilisin the IMC domain 35-fold than the wild-type the between the of Glu112 and the of the residues within the IMC domain are important for binding of the IMC with the protease domain. This does not the binding of the subtilisin inhibitor with the protease domain Based on the data in this IMC-subtilisin and similar folding It by and S. P. F. P. Biochemistry. 1993; 32: PubMed Scopus (84) Google Scholar) that the IMC domain is added in the rate-limiting step of folding is the formation of the IMC-subtilisin Our results to be with this However, because of the between the IMC domain and the the of formation may be higher in the that stabilize the IMC-subtilisin may not be for the of folding. the Glu112 with the of the IMC domain is not for initiating protein folding and does not to be involved in the However, the hydrogen between Glu112 and the IMC is important for autoprocessing In the absence of this the IMC domain and the between the of the IMC domain and the in is for As a result the precursor its IMC domain than Upon from the of the IMC domain remains with the protease domain. This to be for the inhibitory functions of the IMC domain. The dramatically the binding of the IMC domain with subtilisin by E112A-subtilisin displays similar activities peptide and an subtilisin inhibitor that is similar to that with wild-type subtilisin.