Minimizing nonproductive substrate binding: a new look at glucoamylase subsite affinities.
A subsite model as proposed by Hiromi [Hiromi, K. (1970) Biochem. Biophys. Res. Commun. 40, 1-6] has been applied to various hydrolases including glucoamylase (GA). The model assumes a single enzyme complex, a hydrolytic rate constant which is independent of substrate length, and a ratelimiting hydrolytic step. Recent kinetic studies with GA contradict these assumptions. Here we reevaluate the substrate binding of GA studying the pre-steady-state kinetics with glucose, which is reported here for the first time, and maltose. The association equilibrium constants for glucose and maltose interactions with wild-type and Trp120-->Phe GA from Aspergillus awamori in H2O and D2O buffers were obtained. Kinetic results indicate that a single glucose molecule binds to GA weakly by a single-step mechanism, E + G1<-->EG1, under the conditions studied. Similar fluorescence intensities of the GA-glucose and GA-maltose complexes, the high tryptophan concentration around subsite 1, crystal structures of various inhibitor complexes, pre-steady-state and steady-state modeling, feasibility of condensation reactions, and other evidence strongly suggest that glucose binds at subsite 1. These results conflict with the high subsite 2 and low subsite 1 affinities obtained using Hiromi's model. Using the substrate association constants for glucose and maltose obtained by pre-steady-state kinetics, the affinity of alpha-glucose for subsite 1 is shown to be substantially higher than the apparent affinity of glucose for subsite 2. We propose a GA catalytic mechanism whereby substrate binding is initiated by subsite 1 interactions with the nonreducing end of the oligosaccharide substrate, minimizing nonproductive substrate binding. Through conformational changes, entropic contributions, and increased local concentration, subsite 2 subsequently has enhanced affinity for the second covalently linked glucosyl residue.
Natarajan
S K
SK
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County 21250, USA.
Sierks
M R
MR
D001230:Aspergillus / Q000201:enzymology; D001665:Binding Sites; D017022:Flow Injection Analysis; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D008320:Maltose; D005087:Glucan 1,4-alpha-Glucosidase; D005947:Glucose