Composition, N-terminal amino acids, and chain length of a β-glucosidase from Aspergillus wenth

Legler, Günter; Radloff, Michael; Kempfle, M.

doi:10.1016/0005-2795(72)90252-8

Cited by 17 publications

(7 citation statements)

References 24 publications

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“…The labile ,3-glucopyranosyl fluoride contained maximally 8 % free fluoride. (Legler et al, 1972) was a gift from Professor G. Legler (Institut fur Biochemie der Universitat zu K6ln, K6ln, Germany). Pig kidney trehalase (lot no.…”

Section: Methodsmentioning

confidence: 99%

The interaction of 1-fluoro-d-glucopyranosyl fluoride with glucosidases

Konstantinidis

Sinnott

1991

Biochemical Journal

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1. 1-Fluoro-D-glucopyranosyl fluoride undergoes pH-independent loss of F- ion at a rate of 1 x 10(-8) s-1 at 50.0 degrees C, some 10(3)-fold slower than alpha-D-glucopyranosyl fluoride and 4 x 10(4)-fold slower than beta-D-glucopyranosyl fluoride. 2. The (inverting) amyloglucosidase II of Aspergillus niger hydrolyses the difluoride according to Michaelis-Menten kinetics (Km 34 mM and kcat. 0.27 s-1), by apparently the same (simple) mechanism by which it hydrolyses alpha-D-glucopyranosyl fluoride (Km 38 mM and kcat. 730 s-1), rather than by the Hehre resynthesis-hydrolysis mechanism used to transform beta-D-glucopyranosyl fluoride. 3. The difluoride is also a substrate for the (inverting) trehalase of pig kidney [Km 17.3 mM and Vmax. 6.2 x 10(-4) relative to alpha-D-glucopyranosyl fluoride (Km 38 mM]). 4. The quantitatively similar effect of fluorine substitution on the one-step enzymic reactions and on the non-enzymic reactions suggests that they go through similar (oxocarbonium-ion-like) transition states. 5. The difluoride is a substrate for the (retaining) beta-glucosidases from Aspergillus wentii (A3 enzyme) and sweet-almond meal (B isoenzyme) and for the retaining alpha-glucosidase from rice: comparison with the appropriate monofluoride reveals a variable rate-retarding effect of the second fluorine atom on kcat./Km that correlates with other measures of oxocarbonium ion character in the transition state. 6. The difluoride is a substrate for the (retaining) alpha-glucosidase from yeast, but also gives an insidious mimicry of active-site-directed irreversible inhibition, which we tentatively attribute either to formation of the non-covalent complex or to the fluoroglucosyl-enzyme increasing the well-known tendency of this enzyme to come out of solution by adsorption on the walls of the vessel.

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Section: Methodsmentioning

confidence: 99%

The interaction of 1-fluoro-d-glucopyranosyl fluoride with glucosidases

Konstantinidis

Sinnott

1991

Biochemical Journal

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“…wentii (Rohm GmbH, Darmstadt, F.R.G.) as described [3]. Its specific activity with 20 mM p-nitrophenyl-fl-D-glucospyranoside at pH 4.0 and 35'C was 32 U/mg protein (30 U/mg had been found previously).…”

Section: Elzqmiementioning

confidence: 99%

“…The following kinetic constants were found with the 4-methylumbelliferyl glucoside at pH 4.0 and 25 T : K, = 0.57 mM, V = 123 U/mg. Protein concentrations were determinated by ultraviolet absorption with A: 78 mgflm' = 1.91 cm-' [3]. Details of the inhibition and rqctivation kinetics are given in the legends to the figures.…”

Section: Elzqmiementioning

confidence: 99%

“…The binding site was identified (by isolation and structure determination of a radioactive peptide) as the same aspartate residue that had been labeled with the active-site-directed inhibitor, conduritol B epoxide, in a previous study. P-D-Glucosidase A3, isolated from culture filtrates of Aspergillus wentii, has been characterized with respect to its enzymatic [1,2] and molecular properties [3]. Two carboxyl groups are thought to participate in the catalytic action; one of them has been identified as an aspartic acid residue by labeling the enzyme with the active-site-directed inhibitor conduritol B epoxide [4].…”

mentioning

confidence: 99%

“…P-D-Glucosidase A3, isolated from culture filtrates of Aspergillus wentii, has been characterized with respect to its enzymatic [1,2] and molecular properties [3]. Two carboxyl groups are thought to participate in the catalytic action; one of them has been identified as an aspartic acid residue by labeling the enzyme with the active-site-directed inhibitor conduritol B epoxide [4].…”

mentioning

confidence: 99%

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Reaction of β‐d‐Glucosidase A₃ from Aspergillus wentii with d‐Glucal

Legler

Roeser

Illig

1979

European Journal of Biochemistry

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D-Glucal acts as mixed competitive/non-competitive inhibitor against 1-D-glucosidase A3 from Asprypillus wentii with slow approach to the steady-state rate of substrate hydrolysis. The same rate is reached whether D-glucal competes directly with the substrate or the substrate displaces D-glucal that has been bound by preincubation in the absence of substrate. The Ki for competitive inhibition and the rate constants for the approach to the steady state are in agreement with the kinetic constants for the hydration of D-glucal to 2-deoxy-~-glucose.The concentration dependence of the inhibition shows that one molecule of D-glucal (I) binds with complete inhibition but the biphasic dissociation kinetics of the enzyme-glucal complex and labeling studies point to an additional binding site. An E12 complex can be isolated at pH 6 and 0 "C by rapid ion-exchange chromatography. This complex can be reactivated at pH 4 and room temperature ; all the D-glucal is released as 2-deoxy-~-glucose.After denaturation of the El2 complex one molecule of D-glucal remains bound to the enzyme. The binding site was identified (by isolation and structure determination of a radioactive peptide) as the same aspartate residue that had been labeled with the active-site-directed inhibitor, conduritol B epoxide, in a previous study. P-D-Glucosidase A3, isolated from culture filtrates of Aspergillus wentii, has been characterized with respect to its enzymatic [1,2] and molecular properties [3]. Two carboxyl groups are thought to participate in the catalytic action; one of them has been identified as an aspartic acid residue by labeling the enzyme with the active-site-directed inhibitor conduritol B epoxide [4]. In order to obtain more information about the nature of the active site other inhibitors were investigated. In addition to cationic fi-glucosyl derivatives, we found D-glucal a promising candidate for further studies. The half-chair conformation of D-glucal (I) resembles the conformation of the glucosyl cation (11) which is supposed to be an intermediate in the enEnzyme. /-i>-Glucoside glucohydrolase (EC 3.2.1.21) zymatic hydrolysis of glucosides. It might, therefore, be considered as transition state analog, provided the missing hydroxyl group and the different location of the double bond do not seriously interfere with the binding process. Inhibitors resembling the transition state bind much better than direct analogs of substrates [6]. Some inhibitor dissociation constants for P-glucosidase A3 are: 1-D-glucose 2.8 mM, p-nitrophenyl-P-D-1 -thioglucoside 0.9 mM, but D-gluconod-lactone (111) 0.013 mM. In additon, D-ghCd might be able to form a covalent bond with a nucleophilic group at the active site.Literature data show that these considerations are only partially correct. The inhibition of 8-galactosidase from Escherichia coli by D-galactal is of similar strength to the inhibition by D-galactono-&lactone but the approach to the enzyme-inhibitor equilibrium is a slow process [7]. The inhibitor is released as 2-deoxy-D-galactose which...

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