Active site similarities of glucose dehydrogenase, glucose oxidase, and glucoamylase probed by deoxygenated substrates

Michael Sierks, Klaus Bock, Susanne Refn, Birte Svensson

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

The specificity constants, kcat/KM, were determined for glucose oxidase and glucose dehydrogenase using deoxy-D-glucose derivatives and for glucoamylase using deoxy-D-maltose derivatives as substrates. Transition-state interactions between the substrate intermediates and the enzymes were characterized by the observed kcat/Km values and found to be very similar. The binding energy contributions of individual sugar hydroxyl groups in the enzyme/substrate complexes were calculated using the relationship Δ(ΔG) = -RT ln [(kcat/KM)deoxy/(kcat/K M)hydroxyl] for the series of analogues. The activity of all three enzymes was found to depend heavily on the 4- and 6-OH groups (4′- and 6′-OH in maltose), where changes in binding energies from 10 to 18 kJ/mol suggested strong hydrogen bonds between the enzymes and these substrate OH groups. The 3-OH (3′-OH in maltose) was involved in weaker interactions, while the 2-OH (2′-OH in maltose) had a very small if any role in transition-state binding. The three enzyme-substrate transition-state interactions were compared using linear free energy relationships (Withers, S. G., & Rupitz, K. (1990) Biochemistry 29, 6405-6409) in which the set of kcat/KM values obtained with substrate analogues for one enzyme is plotted against the corresponding values for a second enzyme. The high linear correlation coefficients (p) obtained, 0.916, 0.958, and 0.981, indicate significant similarity in transition-state interactions, although the three enzymes lack overall sequence homology. A short amino acid sequence, however, which is critical for glucoamylase activity (Clarke, A. J., & Svensson, B. (1984) Carlsberg Res. Commun. 49, 559-566; Sierks, M. R., Ford, C., Reilly, P. J., & Svensson, B. (1989) Protein Eng. 2, 621-625) was recognized in glucose oxidase. Whether this area also plays a crucial role in that enzyme is not yet known.

Original languageEnglish (US)
Pages (from-to)8972-8977
Number of pages6
JournalBiochemistry
Volume31
Issue number37
StatePublished - 1992
Externally publishedYes

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Glucose 1-Dehydrogenase
Glucan 1,4-alpha-Glucosidase
Glucose Oxidase
Catalytic Domain
Substrates
Enzymes
Maltose
Binding energy
Hydroxyl Radical
Derivatives
Biochemistry
Sequence Homology
Sugars
Free energy
Hydrogen
Amino Acid Sequence
Hydrogen bonds

ASJC Scopus subject areas

  • Biochemistry

Cite this

Active site similarities of glucose dehydrogenase, glucose oxidase, and glucoamylase probed by deoxygenated substrates. / Sierks, Michael; Bock, Klaus; Refn, Susanne; Svensson, Birte.

In: Biochemistry, Vol. 31, No. 37, 1992, p. 8972-8977.

Research output: Contribution to journalArticle

Sierks, Michael ; Bock, Klaus ; Refn, Susanne ; Svensson, Birte. / Active site similarities of glucose dehydrogenase, glucose oxidase, and glucoamylase probed by deoxygenated substrates. In: Biochemistry. 1992 ; Vol. 31, No. 37. pp. 8972-8977.
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