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

S Becker,D Palm,R Schinzel

doi:10.1016/s0021-9258(17)41971-5

Abstract

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.

Highlights

Substrate analogs are used in combination witshite- It is assumed thatglycogen or linear polysaccharide chains directed mutagenesis to probe specific interactions be- can bind to phosphorylase in two distinct modes: one in which tween substrate and enzyme in the forward and revethreseterminalglucosyl residue is bound in a positionsuitable for direction of the Escherichia coli maltodextrin phospho- phosphorolysis(phosphorolysis mode, P-mode) and one in rylasereaction
In thephosphorolysis(degradation) which the terminalglucosyl residue isbound in a position that mode, removal of the 2-OH group of the terminal glucaollsoews it to act as a glucosyl acceptor of the polysaccharide results in a 30-fold redoufcKti,on (French and Wild, 1953; Chao et al 1969; Gold et al 1974; while similar changes were of no influence when the Segel, 1975)
Theanswertothequestion act with the terminal glucose residuAe.2-fold increase whetherbinding of oligosaccharides inthe phosphorolysis in rate was observed in both directions using the 2-de-mode is differentfrombinding in the synthesis mode on a oxy derivatives

Summary

Dissecting Differential Binding in the Forward and Reverse

Reaction of Escherichia coli Maltodextrin Phosphorylase Using 2-Deoxyglucosyl Substrates*. Enzyme Purification-Wild-type and mutant enzyme were purified as described (Schinzeland Palm, 1990)with an additional fast protein detection of differences in binding of substrates inthe forward and reverse reaction. Such differences would be a strong indication for structural rearrangements at the active site during catalysis. From the crystal structure of the rabbit skeletal muscle enzyme and its complexes with glucose and Glc-1-P derivatives, two active site residues (lj76'73 and G ~ u ~h~ad' )been assigned which are involved in interactions between the 2-OH group of the glucose residue and theenzyme (Johnson et al, 1990; Martin et al 1990).In aparallel approach, we mapped functionally important amino acid residues of the Escherichia coli enzyme by site-directedmutagenesis and kinetic studies.

MATERIALS AND METHODS

DISCUSSION

Glu n