Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens

D. Becker, K. Johnson, Anu Koivula, M. Schulein, M. Sinnott (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.

Original languageEnglish
Pages (from-to)315 - 319
Number of pages5
JournalBiochemical Journal
Volume345
Issue number2
DOIs
Publication statusPublished - 2000
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Hydrolysis
Fluorides
Enzymes
Cellobiose
Kinetics
Glycoside Hydrolases
cellobiosyl fluoride
Catalytic Domain
Crystal structure
Chemical activation
Nuclear magnetic resonance
Impurities
Monitoring
Substrates

Cite this

@article{31951ea29f4e4a1bbbe301f118076946,
title = "Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens",
abstract = "We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Sch{\"u}lein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0{\%} of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.",
author = "D. Becker and K. Johnson and Anu Koivula and M. Schulein and M. Sinnott",
year = "2000",
doi = "10.1042/bj3450315",
language = "English",
volume = "345",
pages = "315 -- 319",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland press",
number = "2",

}

Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens. / Becker, D.; Johnson, K.; Koivula, Anu; Schulein, M.; Sinnott, M. (Corresponding Author).

In: Biochemical Journal, Vol. 345, No. 2, 2000, p. 315 - 319.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens

AU - Becker, D.

AU - Johnson, K.

AU - Koivula, Anu

AU - Schulein, M.

AU - Sinnott, M.

PY - 2000

Y1 - 2000

N2 - We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.

AB - We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.

U2 - 10.1042/bj3450315

DO - 10.1042/bj3450315

M3 - Article

VL - 345

SP - 315

EP - 319

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 2

ER -