Klassifizierung der Pullulanase als Exoenzym mit Hilfe der Gaschromatographie<sup>1</sup>

Wallenfels, Kurt; Rached, I. R.; Hucho, Ferdinand

doi:10.1111/j.1432-1033.1969.tb19596.x

Cited by 13 publications

(7 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Subsequently the reverse reaction takes place thereby yielding either the a-or #?-pyranose or the a-or #?-furanose. Since the velocity of the ring closure is greater than the velocity of the ring opening by the factor lo4 [28] virtually no acyclic open aldehyde dissociates from the active center as has been shown by our polarographic experiments [5].…”

Section: The Mechanism Of the Enzymatically Catalyzed Mutarotationsupporting

confidence: 77%

“…Earlier investigations had shown that the enzymatic catalysis of mutarotation by mutarotase from appears to follow a bifunctional mechanism as well [5]. I n this paper we are now presenting a detailed investigation of the mechanism of action of the functional groups of this enzyme.…”

mentioning

confidence: 85%

“…This experiment, therefore, is proof of the assumption first proposed by Lowry [7] that the openchain aldehyde is a necessary step in mutarotation. Polarographic experiments on the other hand had shown that the open-chain aldehyde does not leave the active center of the mutarotase [5]. The cyclisation to the other anomer or to the furanose takes place on the enzyme.…”

Section: Escherichk Coli Mutarotasementioning

confidence: 99%

See 2 more Smart Citations

The Enzymatically Catalyzed Mutarotation. The Mechanism of Action of Mutarotase (Aldose 1-Epimerase) from Escherichia coli

Hucho

Wallenfels

1971

Eur J Biochem

View full text Add to dashboard Cite

1. The free energy, enthalpy, and entropy of activation of the enzymatically catalyzed mutarotation of glucose have been determined: AG* 11.9 kcal/mol, AH* 12.8 kcal/mol, AS* 3.1 cal x mol-l x K-l. 2.The pH-dependence of the enzymatic activity has been investigated. The active center of the mutarotase from Escherichiu coli contains one functional group (pK 5.5) which in the dissociated form has only a catalytic function, and a second one (pK 7.6) which is responsible for the binding of the substrate.3. The binding group of the substrate has been determined. By comparing the IMichaelis and inhibitor constants of a variety of substrates and inhibitors it is concluded that the substrate is bound to the enzyme over an equatorial hydroxyl group a t C-2 of the carbohydrate ring.4. The products of the enzymatically catalyzed mutarotation have been analyzed by gas chromatography. These experiments show that the mutarotase catalyzes not only the anomerisation but also the ring isomerisation. The result is interpreted as evidence of an acyclic intermediate of the mutarotation of the aldoses.5 . The results are summarized in a reaction mechanism of the enzymatically catalyzed mutarotation. Its characteristic feature is that it describes the action of the mutarotase from E. coli as a base catalysis.The interpretation of the enzymatic action as a bifunctional catalysis, i.e. a simultaneous attack of an electrophilic and a nucleophilic group of the enzyme on the substrate, represents a preferred concept in today's enzymology. It is based in part on experiments dealing with the (nonenzymatic) catalysis of the mutarotation of glucose [1,2]. Therefore, in the enzymological literature mutarotation is considered to be a model reaction (for example [3,4] &-D-glucose r=---==\ /?-D-glucose(1) appears to follow a bifunctional mechanism as well [5]. I n this paper we are now presenting a detailed investigation of the mechanism of action of the functional groups of this enzyme. We provide an answer to the question in which way they are involved in the catalysis: by binding the substrate or by directly participating in one or more of the catalytic steps. It was of special interest to investigate the relationship of the mutarotase to the other (nonenzymatic) catalysts of the mutarotation. Even before the concept of bifunctional catalysis the related concept of acid-base catalysis has been developed [6], again partly based on investigations of the mutarotation. By analyzing the pH and temperature dependence of the kinetic parameters we investigated the applicability of the two concepts for the enzymatically catalyzed mutarotation and its relationship to the nonenzymatic catalysis of mutarotation.An unsettled problem of mutarotation is the hypothesis of an acyclic aldehyde intermediate [7 -91. By chromatographic analysis of the reaction products during mutarotation we present evidence that the enzymatically catalyzed mutarotation proceeds via the open chain form of the aldoses.

show abstract

Section: The Mechanism Of the Enzymatically Catalyzed Mutarotationsupporting

confidence: 77%

mentioning

confidence: 85%

Section: Escherichk Coli Mutarotasementioning

confidence: 99%

See 1 more Smart Citation

The Enzymatically Catalyzed Mutarotation. The Mechanism of Action of Mutarotase (Aldose 1-Epimerase) from Escherichia coli

Hucho

Wallenfels

1971

Eur J Biochem

View full text Add to dashboard Cite

show abstract

“…The availability of highly purified preparations of pullulanase from Aerobacter aerogenes [5] has made this enzyme invaluable in the analysis of the fine structure of amylopectin [6]. Pullulanase, however, is of limited use in the analysis of glycogen structure because, although it is able to hydrolyse some branch linkages in degraded glycogen, it has little or no action on the undegraded macromolecule [7].…”

Section: Introductionmentioning

confidence: 99%

A glycogen‐debranching enzyme from Cytophaga

et al. 1970

View full text Add to dashboard Cite

“…in 5 ml of 0.01 M acetate buffer, pH 5.4, was added 2 ml of Aerobacter pullulanase 6 ) containing 500 total units of activity, and a portion of the digest incubated for 24 hr at 40°C was subjected to chromatography on a column of Sephadex G-75 (¢ 2.0 x 55 cm) in which the dextrins produced were separated into two peak fractions: one was eluted at around the void volume zone of the column with a yield of 70%, and the other significantly later with a yield of 30 %. The former fraction seemed to be a mixture of slight hydrolysis products of the ,B-limit dextrin.…”

mentioning

confidence: 99%