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 language | English (US) |
|---|---|
| Pages (from-to) | 2147-2153 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 261 |
| Issue number | 5 |
| State | Published - 1986 |
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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 journal › Article
}
TY - JOUR
T1 - Catalytic flexibility of glycosylases. The hydration of maltal by β-amylase to form 2-deoxymaltose
AU - Hehre, E. J.
AU - Kitahata, S.
AU - Brewer, Curtis F.
PY - 1986
Y1 - 1986
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.
AB - 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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M3 - Article
C2 - 2418022
AN - SCOPUS:0022999833
VL - 261
SP - 2147
EP - 2153
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 5
ER -