Inhibition of Glucoamylases from a Rhizopus Sp. and Aspergillus saitoi by Aminoalcohol Derivatives

Abstract

The mechanism of inhibition of the two glucoamylases from a Rhizopus sp. and Aspergillus saitoi by aminoalcohol derivatives was investigated. Hydrolysis of maltose by the glucoamylases was inhibited competitively by aminoalcohols at pH 5.0, and tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1,3-propanediol and 2-aminocyclohexanol were relatively good inhibitors of the glucoamylases among the aminoalcohol derivatives tested. One hydroxyl group and an amino group in these inhibitors were indispensable for the inhibitory action, and the addition of other hydroxyl, amino or ethyl groups was enhancing. With an increase in pH from 4.0 to 6.0, the Ki values of the aminoalcohols decreased. This result suggested the participation of a carboxyl group, which was related to the glucoamylase activity and had a pKa of 5.7, in the binding of aminoalcohols. The UV difference spectra induced on binding of the aminoalcohol analogues with the glucoamylases may indicate a change of the environment of tryptophan residues to a slightly higher pH on inhibitor binding. The influence of aminoalcohols on the fluorescence intensity due to tryptophan residues and the CD-spectra of the glucoamylases was less than that of maltitol. Thus, the interaction of aminoalcohols with tryptophan residues in the glucoamylases might be less pronounced than that in the case of substrate analogues. The modes of binding of the aminoalcohols with the two glucoamylases were very similar. Therefore, the phenomenon might be a common feature of glucoamylases in general.

Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers

“…PP i has been suggested to be a universal regulator of many cell processes [35]. It is thus not unlikely that the reported effects of Tris on other enzymes and more complex biological systems that do not utilize PP i as substrate [36–40] are explained by Tris competing with PP i as regulator.…”

Structure of the catalytic domain of glucoamylase fromAspergillus niger

“…The average B factor for the glycerol molecule is 46.7 Å 2 . The presence of Tris in the active site is not surprising as Tris is a well known inhibitor of glycosidases, including glucoamylases (Iwama et al, 1985;James & Lee, 1996). Tris acquired during purification has also been found in the active site of a yeast glucoamylase from Saccharomycopsis fibuligera (PDB codes 1ayx and 2fba;Š evčík et al, 1998.…”

“…Such electrostatic charge stabilization is an important element that is exploited by many glucoamylase inhibitors. One such inhibitor is 1-deoxynojirimycin, which has an endocyclic N atom in place of O (Harris et al, 1993), and Tris appears to exploit a similar interaction to confer enzyme inhibition (Iwama et al, 1985).…”

“…The structure of the catalytic domain reveals the active site in complex with Tris and glycerol at the À1 and +1 subsites, respectively. While Tris is a well known inhibitor of glucoamylases (Iwama et al, 1985;James & Lee, 1996), its interactions with the active site of an Aspergillus glucoamylase have not been structurally characterized. In our structure, the Tris and glycerol molecules tightly associate with the active site by closely mimicking substrate hydrogen-bonding interactions, while the amino group of Tris appears to be stabilized by a direct hydrogen bond to the catalytic residue Glu203(179).…”

Computational studies of glucoamylase selectivity

“…Partial or total proteolytic excision of the SBD leads to enzyme forms able to de grade solubilized starch but not its raw or granular form. Extensive characterization of both GAs has been made through the years (Takahashi et al, 1985;S vensson et al, 1986; but more recent efforts to modify GA properties to better suit industrial uses require a better knowledge of GA struc ture/function relationships.…”