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


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 UniProtKB Entries

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