Investigation of the role of a second conserved serine in carboxylesterases via site-directed mutagenesis

Jeanette E. Stok, Andrey Goloshchapov, Cheng Song, Craig E. Wheelock, Maher B H Derbel, Christophe Morisseau, Bruce D. Hammock

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Carboxylesterases are enzymes that catalyze the hydrolysis of ester and amide moieties. These enzymes have an active site that is composed of a nucleophile (Ser), a base (His), and an acid (Glu) that is commonly known as a catalytic triad. It has previously been observed that the majority of carboxylesterases and lipases contain a second conserved serine in their active site [Proteins, 34 (1999) 184]. To investigate whether this second serine is also involved in the catalytic mechanism, it was mutated to an alanine, a glycine or a cysteine. Site-directed mutagenesis of this conserved serine resulted in a loss of specific activity, in both the S247G and S247A mutants (5- to 15-fold), which was due to a decrease in the rate of catalysis (k cat). Due to the instability of the S247C mutant no reliable data could be attained. A carbamate inhibitor, carbaryl, was then employed to investigate whether this decrease in the k cat was due to the rate of formation of the acyl-enzyme intermediate (k 2) or the rate of deacylation (k 3). The S247A mutant was found only to alter k 2 (2.5-fold decrease), with no effect on k 3. Together with information inferred from a human carboxylesterase crystal structure, it was concluded that this serine provides an important structural support for the spatial orientation of the glutamic acid, stabilizing the catalytic triad so that it can perform the hydrolysis.

Original languageEnglish (US)
Pages (from-to)247-255
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume430
Issue number2
DOIs
StatePublished - Oct 15 2004

Keywords

  • Carboxylesterase
  • Catalytic triad
  • Serine
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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    Stok, J. E., Goloshchapov, A., Song, C., Wheelock, C. E., Derbel, M. B. H., Morisseau, C., & Hammock, B. D. (2004). Investigation of the role of a second conserved serine in carboxylesterases via site-directed mutagenesis. Archives of Biochemistry and Biophysics, 430(2), 247-255. https://doi.org/10.1016/j.abb.2004.06.020