Probing pH-dependent functional elements in proteins: Modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A

Gerd Wohlfahrt, Tarmo Pellikka, Harry Boer, Tuula Teeri, Anu Koivula (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

35 Citations (Scopus)

Abstract

Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl−carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl−carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl−carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl−carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (Tm) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl−carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior.
Original languageEnglish
Pages (from-to)10095-10103
Number of pages9
JournalBiochemistry
Volume42
Issue number34
DOIs
Publication statusPublished - 2003
MoE publication typeA1 Journal article-refereed

Fingerprint

Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Carboxylic Acids
Enzymes
Proteins
Stabilization
Circular dichroism spectroscopy
Deprotonation
Catalytic Domain
Ultraviolet spectroscopy
Amides
Cellulose
Tryptophan
Catalysis
Protons
Tunnels
Hydrogen bonds
Fluorescence
Geometry
Substrates

Keywords

  • carboxyl group
  • Trichoderma reesei
  • protein engineering
  • proteins
  • hydrogen bonds
  • protein stability
  • stability
  • fungal cellulase
  • cellulase

Cite this

@article{8392aa7db7a9445ca9903bc478af9cd4,
title = "Probing pH-dependent functional elements in proteins: Modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A",
abstract = "Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl−carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl−carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl−carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl−carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (Tm) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl−carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior.",
keywords = "carboxyl group, Trichoderma reesei, protein engineering, proteins, hydrogen bonds, protein stability, stability, fungal cellulase, cellulase",
author = "Gerd Wohlfahrt and Tarmo Pellikka and Harry Boer and Tuula Teeri and Anu Koivula",
year = "2003",
doi = "10.1021/bi034954o",
language = "English",
volume = "42",
pages = "10095--10103",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society ACS",
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}

Probing pH-dependent functional elements in proteins : Modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A. / Wohlfahrt, Gerd; Pellikka, Tarmo; Boer, Harry; Teeri, Tuula; Koivula, Anu (Corresponding Author).

In: Biochemistry, Vol. 42, No. 34, 2003, p. 10095-10103.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Probing pH-dependent functional elements in proteins

T2 - Modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A

AU - Wohlfahrt, Gerd

AU - Pellikka, Tarmo

AU - Boer, Harry

AU - Teeri, Tuula

AU - Koivula, Anu

PY - 2003

Y1 - 2003

N2 - Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl−carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl−carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl−carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl−carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (Tm) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl−carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior.

AB - Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl−carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl−carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl−carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl−carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (Tm) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl−carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior.

KW - carboxyl group

KW - Trichoderma reesei

KW - protein engineering

KW - proteins

KW - hydrogen bonds

KW - protein stability

KW - stability

KW - fungal cellulase

KW - cellulase

U2 - 10.1021/bi034954o

DO - 10.1021/bi034954o

M3 - Article

VL - 42

SP - 10095

EP - 10103

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 34

ER -