Functional tuning of the catalytic residue pKa in a de novo designed esterase

Katharina Hiebler, Zsófia Lengyel, Carlos A. Castañeda, Olga V. Makhlynets

Research output: Research - peer-reviewArticle

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Abstract

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p-nitrophenyl acetate (pNPA) and 4-nitrophenyl-(2-phenyl)-propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single-histidine residue (His144) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pKa value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pKa calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pKa value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pKa value of this residue in water. We determined that a short-range favorable interaction with Glu127 contributes to the elevated pKa of His144. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pKa of its active nucleophile, His144, by 0.7 units. As a direct result of the decrease in the His144 pKa value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine-promoted catalysis.

LanguageEnglish (US)
JournalProteins: Structure, Function and Bioinformatics
DOIs
StateAccepted/In press - 2017

Fingerprint

Esterases
Histidine
Tuning
Electrostatics
Static Electricity
Calmodulin
Nuclear magnetic resonance
Enzymes
Nucleophiles
Propionates
Catalysis
Spectrometers
Ions
Calcium
Water
Experiments
4-nitrophenyl acetate
4-nitrophenyl

Keywords

  • Calmodulin
  • Catalysis
  • Electrostatic potential
  • Ester hydrolysis
  • NMR
  • PH rate profile
  • pKa of histidine

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

Cite this

Functional tuning of the catalytic residue pKa in a de novo designed esterase. / Hiebler, Katharina; Lengyel, Zsófia; Castañeda, Carlos A.; Makhlynets, Olga V.

In: Proteins: Structure, Function and Bioinformatics, 2017.

Research output: Research - peer-reviewArticle

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