Contribution of residues in the reactive site loop of chymotrypsin inhibitor 2 to protein stability and activity.


Abstract

Residues in the active site loop of the serine protease inhibitor, chymotrypsin inhibitor 2, thought to play an important role in loop stability and inhibitory activity, have been investigated by site-directed mutagenesis. Substitutions at residues 58 (threonine in wild type) and 60 (glutamic acid in wild type), which flank the scissile bond (Met-59-Glu-60) and are conserved among the potato inhibitor I family of serine protease inhibitors, are found to be of some importance in the global stability of the protein, as measured by guanidinium chloride-induced denaturation, but are essential for its inhibitory activity. Mutation of either Thr-58 or Glu-60 to alanine results in a decrease in stability of 0.7 +/- 0.1 kcal mol-1. These values reflect the loss of hydrogen bonds between the hydroxyl group of Thr-58 with Glu-60 and Arg-67 and hydrogen bonds and a salt bridge between Glu-60 and Arg-62 and Arg-65. In addition, these mutants were found to be much weaker inhibitors of the serine protease subtilisin BPN'. The dissociation constants for inhibition, Ki, were found to be (7.0 +/- 0.4, 540 +/- 30, and 980 +/- 50) x 10(-13) M, for wild type, T58A, and E60A, respectively. Further, we find that these mutants are only temporary inhibitors of subtilisin BPN', unlike wild type. Over long time scales, we observe a reversal of inhibition because of hydrolysis of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 1863, 1864, 1865, 1866, 1867, 1868 Extra Details: dG and ddG were measured in the presence of [GdnHCl]50% chymotrypsin inhibitor 2; site-directed mutagenesis; activity;,hydrogen bonds; protein stability; salt bridge

Submission Details

ID: bGtwqygw3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:18 p.m.

Version: 1

Publication Details
Jackson SE;Fersht AR,Biochemistry (1994) Contribution of residues in the reactive site loop of chymotrypsin inhibitor 2 to protein stability and activity. PMID:7947796
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3CI2 1993-10-31 REFINEMENT OF THE THREE-DIMENSIONAL SOLUTION STRUCTURE OF BARLEY SERINE PROTEINASE INHIBITOR 2 AND COMPARISON WITH THE STRUCTURES IN CRYSTALS
1CIS 1993-10-31 CONTEXT DEPENDENCE OF PROTEIN SECONDARY STRUCTURE FORMATION. THE THREE-DIMENSIONAL STRUCTURE AND STABILITY OF A HYBRID BETWEEN CHYMOTRYPSIN INHIBITOR 2 AND HELIX E FROM SUBTILISIN CARLSBERG
1CIR 1996-01-29 COMPLEX OF TWO FRAGMENTS OF CI2 [(1-40)(DOT)(41-64)]
1LW6 2002-08-21 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1YPC 1994-01-31 1.7 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
1CQ4 1998-11-25 1.8 CI2 MUTANT WITH TETRAGLUTAMINE (MGQQQQGM) REPLACING MET59
5FFN 2016-05-18 1.8 Complex of subtilase SubTY from Bacillus sp. TY145 with chymotrypsin inhibitor CI2A
5FBZ 2016-05-18 1.9 Structure of subtilase SubHal from Bacillus halmapalus - complex with chymotrypsin inhibitor CI2A
1YPA 1994-01-31 2.0 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
1YPB 1994-01-31 2.0 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
2CI2 1988-09-07 2.0 CRYSTAL AND MOLECULAR STRUCTURE OF THE SERINE PROTEINASE INHIBITOR CI-2 FROM BARLEY SEEDS
2SNI 1988-09-07 2.1 STRUCTURAL COMPARISON OF TWO SERINE PROTEINASE-PROTEIN INHIBITOR COMPLEXES. EGLIN-C-SUBTILISIN CARLSBERG AND CI-2-SUBTILISIN NOVO
1COA 1994-01-31 2.2 THE EFFECT OF CAVITY CREATING MUTATIONS IN THE HYDROPHOBIC CORE OF CHYMOTRYPSIN INHIBITOR 2
1CIQ 1996-03-08 2.2 COMPLEX OF TWO FRAGMENTS OF CI2, RESIDUES 1-40 AND 41-64

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.6 Subtilisin-chymotrypsin inhibitor-2A P08626 ICI3_HORVU
100.0 Subtilisin-chymotrypsin inhibitor-2A P01053 ICI2_HORVU