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

Biochemistry. 1994 Nov 22;33(46):13880-7. doi: 10.1021/bi00250a042.

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)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aspartic Acid / metabolism
  • Binding Sites
  • Chymotrypsin / antagonists & inhibitors*
  • Glutamic Acid / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Mutation
  • Peptides
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Protein Denaturation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Solanum tuberosum
  • Subtilisins / metabolism
  • Thermodynamics
  • Threonine / metabolism

Substances

  • Peptides
  • Plant Proteins
  • chymotrypsin inhibitor 2
  • Threonine
  • Aspartic Acid
  • Glutamic Acid
  • Subtilisins
  • Chymotrypsin