HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Becker, S. Articles by Schinzel, R. Search for Related Content PubMed PubMed Citation Articles by Becker, S. Articles by Schinzel, R. Social Bookmarking                      What's this?

J. Biol. Chem., Vol. 269, Issue 4, 2485-2490, 01, 1994

Dissecting differential binding in the forward and reverse reaction of Escherichia coli maltodextrin phosphorylase using 2-deoxyglucosyl substrates

S Becker, D Palm and R Schinzel
Theodor-Boveri-Institut fur Biowissenschaften (Biozentrum), Physiologische Chemie I, Am Hubland, Wurzburg, Germany.

Substrate analogs are used in combination with site-directed mutagenesis to probe specific interactions between substrate and enzyme in the forward and reverse direction of the Escherichia coli maltodextrin phosphorylase reaction. In the phosphorolysis (degradation) mode, removal of the 2-OH group of the terminal glucose of the polysaccharide results in a 30-fold reduction of Km while similar changes were of no influence when the same polysaccharide was used for priming the synthesis. Mutation of active site residues Glu637 or Tyr538 does not change apparent affinity of substrates during degradation. In the synthesis mode, 2-deoxyglucose-1-P as substrate causes a 2-fold reduction of the wild-type kcat/Km while for the Y538F mutant a approximately 7-fold reduction is observed. In contrast, the mutation of Glu637 to Asp causes a 10-fold increase in kcat/Km. Therefore, different binding sites for the terminal glucose residue of the oligosaccharide and glucose-1-P exist. Glu637 and Tyr538 are part of the glucose-1-P binding site and do not interact with the terminal glucose residue. A 2-fold increase in rate was observed in both directions using the 2-deoxy derivatives. This confirms the role of intrinsic electronic effects in stabilizing the transition state. Uncompetitive substrate inhibition at high concentrations of maltoheptaose in the phosphorolysis direction is explained by inhibitory binding of the sugar in the synthesis mode.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				W. Boos and H. Shuman Maltose/Maltodextrin System of Escherichia coli: Transport, Metabolism, and Regulation Microbiol. Mol. Biol. Rev., March 1, 1998; 62(1): 204 - 229. [Abstract] [Full Text] [PDF]