Subsite structure and ligand binding mechanism of glucoamylase

Abstract

1. The basic concept and outline of the subsite theory were described, which correlates quantitatively the subsite structure (the arrangement of subsite affinities) to the action pattern of amylases in a unified manner. 2. The subsite structures of several amylases including glucoamylase were summarized. 3. In parallel with the theoretical prediction obtained therefrom, the binding subsites of glucose, gluconolactone and linear substrates to Rhizopus glucoamylase were investigated experimentally, by using steady-state inhibition kinetics, difference absorption spectrophotometry, and fluorometric titration. 4. From several lines of evidence, it was concluded that gluconolactone, a transition state analogue, is bound at Subsite 1 (nonreducing end side) where a tryptophan residue is located. 5. The stopped-flow kinetic studies have revealed that all the ligand bindings studied consist of two-step mechanism in which a bimolecular association between the enzyme and a ligand to form a loosely bound complex (EL) followed by the unimolecular isomerization process in which EL converts to the final firmly bound EL complex. For substrates the EL may be the productive complex and the fluorescence of the tryptophan located at Subsite 1 is quenched in their isomerization process, most probably a relocation of ligand to occupy this subsite.

Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

“…In summary, the Trp(120) appears to be exposed in enzyme-substrate complexes comprisingthe binding affinity subsites 1,2, and 3 (9, 11), partially protected in complexes with longer substrates and effectively protected only in the inactive enzyme-acarbose complex (9). Since the hydrolysis occurs between sugar residues number 1 and 2 from the non-reducing end of the substrate (11), the experiments with glucoamylase from A. niger also indicate that the essential Trp(120) is probably located at some distance from the catalytic site. It might, however, be involved in maintaining the structural integrity needed for productive substrate binding and catalysis to occur.…”

“…The presence of the inhibitor acarbose afforded protection of both the enzymatic activity and two tryptophanyl residues. All other ligands examined only protected a single tryptophanyl residue, thought to be located in subsite 1 of the substrate binding region (9,11), and in these cases the resulting derivatives were catalytically inactive (9). Thus, the tryptophan essential for hydrolase activity is oxidized in the latter derivatives and three tryptic peptides (29,30) (120) by NBS in the presence of maltose and the protection of this residue in the G2-acarbose complex.…”

“…Glucoamylase (EC 3.2.1.3) which catalyzes the release of D-glucose from the non-reducing ends of starch and related oligo-and polysaccbarides, has a tryplophanyl residue in subsite ~ of the substrate binding region (11,23). Recently, a second essential tryptophan was found in glucoamylase from Aspergillus niger and these two residues apparently play different roles in the action of the enzyme (9).…”

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

“…In addition, the ability to bind maltose was retained in the derivatives 9 However, in contrast to their distinctly different effects on the unmodified enzyme, acarbose and maltose induced very similar difference spectra for the derivatives (Table II). It may thus be inferred that an exposed carboxyl group is connected with an acarbose-binding region situated outside of the subsites 1 through 3 occupied by maltose (21). Modification of the catalytic site caused a decrease in perturbation at 285-287 nm which may reflect reduced accessibility to both tryptophanyl and tyrosyl chromophores.…”

“…Six consecutive glucosyl residues of a substrate chain can be accomodated at binding subsites and catalytic hydrolysis takes place between subsites 1 and 2 (21). The chemical modification of glucoamylases from Rhizopus sp.…”