Catalytic flexibility of glycosylases. The hydration of maltal by β-amylase to form 2-deoxymaltose

E. J. Hehre, S. Kitahata, Curtis F. Brewer

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Abstract

Crystalline, α-glucosidase-free sweet potato β-amylase was found to catalyze hydration of the enolic bond of maltal (α-D-glucopyranosyl-(1→4)-2-deoxy-D-glucal) to form 2-deoxymaltose (α-D-glucopyranosyl-(1→4)-2-deoxy-D-glucose). The reaction at pH 5.0 showed V(max) 0.082 μmol/min/mg and k(m) 94.5 mM. An exceptionally large solvent deuterium isotope effect, V(H)/V(D) = 8, was observed from pH(pD) 4.2 to 5.4; and at pH(pD) 5.0 the effect was found to be directly related to the mole fraction of 2H. The hydration product, isolated from a β-amylase/maltal digest in acetate-d4/D2O buffer (pD 5.4) was identified through its 1H NMR spectrum as α-D-glucopyranosyl-(1→4)-2-deoxy-D-[2(a)-2H]glucose. β-Amylase in 2H2O thus catalyzes deuteration of the double bond of maltal from a direction opposite that assumed for protonation of the glycosidic oxygen atoms of starch chains and maltosaccharides. This finding confirms the functional flexibility of the enzyme's catalytic groups first demonstrated in studies of the reactions catalyzed with α- and β-maltosyl fluoride (Hehre, E.J., Brewer, C.F., and Genghof, D.S. (1979) J. Biol. Chem. 254, 5942-5950). A possible mechanism of the maltal hydration by β-amylase involves protonation of substrate from above as the first and rate-limiting step, followed by formation of a transient carbonium ion-enzyme intermediate. Although other possible mechanisms cannot be ruled out, it is clear that this hydration reaction differs from reactions catalyzed with amylaceous substrates and with α- and β-maltosyl fluoride. The ability of β-amylase to catalyze different types of reactions with different substrates is discussed with respect to observations with other enzymes that, likewise, strongly support the view (Hehre et al.) that the catalytic groups of glycosylases in general may be functionally flexible beyond requirements of the principle of microscopic reversibility.

Original languageEnglish (US)
Pages (from-to)2147-2153
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number5
StatePublished - 1986

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Amylases
Hydration
Protonation
Fluorides
Substrates
Enzymes
Calcium Gluconate
Glucosidases
Ipomoea batatas
Deuterium
Deoxyglucose
Isotopes
Starch
Buffers
Acetates
Nuclear magnetic resonance
2-deoxymaltose
maltal
Ions
Oxygen

ASJC Scopus subject areas

  • Biochemistry

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Catalytic flexibility of glycosylases. The hydration of maltal by β-amylase to form 2-deoxymaltose. / Hehre, E. J.; Kitahata, S.; Brewer, Curtis F.

In: Journal of Biological Chemistry, Vol. 261, No. 5, 1986, p. 2147-2153.

Research output: Contribution to journalArticle

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N2 - Crystalline, α-glucosidase-free sweet potato β-amylase was found to catalyze hydration of the enolic bond of maltal (α-D-glucopyranosyl-(1→4)-2-deoxy-D-glucal) to form 2-deoxymaltose (α-D-glucopyranosyl-(1→4)-2-deoxy-D-glucose). The reaction at pH 5.0 showed V(max) 0.082 μmol/min/mg and k(m) 94.5 mM. An exceptionally large solvent deuterium isotope effect, V(H)/V(D) = 8, was observed from pH(pD) 4.2 to 5.4; and at pH(pD) 5.0 the effect was found to be directly related to the mole fraction of 2H. The hydration product, isolated from a β-amylase/maltal digest in acetate-d4/D2O buffer (pD 5.4) was identified through its 1H NMR spectrum as α-D-glucopyranosyl-(1→4)-2-deoxy-D-[2(a)-2H]glucose. β-Amylase in 2H2O thus catalyzes deuteration of the double bond of maltal from a direction opposite that assumed for protonation of the glycosidic oxygen atoms of starch chains and maltosaccharides. This finding confirms the functional flexibility of the enzyme's catalytic groups first demonstrated in studies of the reactions catalyzed with α- and β-maltosyl fluoride (Hehre, E.J., Brewer, C.F., and Genghof, D.S. (1979) J. Biol. Chem. 254, 5942-5950). A possible mechanism of the maltal hydration by β-amylase involves protonation of substrate from above as the first and rate-limiting step, followed by formation of a transient carbonium ion-enzyme intermediate. Although other possible mechanisms cannot be ruled out, it is clear that this hydration reaction differs from reactions catalyzed with amylaceous substrates and with α- and β-maltosyl fluoride. The ability of β-amylase to catalyze different types of reactions with different substrates is discussed with respect to observations with other enzymes that, likewise, strongly support the view (Hehre et al.) that the catalytic groups of glycosylases in general may be functionally flexible beyond requirements of the principle of microscopic reversibility.

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