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Covalent bond changes as a driving force in enzyme catalysis

Y Huang1, D W Bolen

  • 1Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston 77555-0647.

Biochemistry
|September 14, 1993
PubMed
Summary

This study quantifies covalent and noncovalent energy changes during alpha-chymotrypsin acylation. It reveals that while acylation is favorable, the covalent furoyl group destabilizes the enzyme, with energy derived from bond changes, not binding.

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Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Protein chemistry

Background:

  • Enzyme acylation involves both covalent and noncovalent interactions.
  • Understanding the energetic contributions of these interactions is crucial for enzyme mechanism elucidation.

Purpose of the Study:

  • To develop a procedure for assessing covalent and noncovalent aspects of alpha-chymotrypsin (alpha-Ct) acylation.
  • To determine the energetic contributions of covalent bond formation and noncovalent interactions during acylation by trifluoroethyl furoate.

Main Methods:

  • Developed a procedure to assess covalent and noncovalent contributions to acylation free energy.
  • Evaluated noncovalent free energy differences by comparing unfolding free energy changes of alpha-chymotrypsin and furoyl-chymotrypsin.
  • Analyzed the overall free energy change for acylation at pH 7.

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Main Results:

  • The overall free energy change for acylation was -4.31 kcal/mol at pH 7.
  • Furoyl-chymotrypsin (F-Ct) was found to be 1.6 kcal/mol less stable than alpha-Ct.
  • Covalent linkage of the furoyl moiety was thermodynamically destabilizing to the enzyme.

Conclusions:

  • The driving force for acylation originates from covalent bond-breaking and bond-making during transesterification, not noncovalent binding.
  • A significant amount of noncovalent free energy is relinquished upon forming the acyl-enzyme.
  • The observed destabilization in F-Ct suggests energy transduction occurs during the conversion of the Michaelis complex to the acyl-enzyme.