Site-directed mutagenesis at the active site Trpl20 of Aspergillus awamori glucoamylase

Abstract

Trp120 of Aspergillus awamori glucoamylase has previously been shown by chemical modification to be essential for activity and tentatively to be located near subsite 4 of the active site. To further test its role, restriction sites were inserted in the cloned A.awamori gene around the Trp120 coding region, and cassette mutagenesis was used to replace it with His, Leu, Phe and Tyr. All four mutants displayed 2% or less of the maximal activity (kcat) of wild-type glucoamylase towards maltose and maltoheptaose. Michaelis constants (KM) of mutants decreased 2- to 3-fold for maltose and were essentially unchanged for maltoheptaose compared with the wild type, except for a greater than 3-fold decrease for maltoheptaose with the Trp120----Tyr mutant. This mutant also bound isomaltose more strongly and had more selectivity for its hydrolysis than wild-type glucoamylase. A subsite map generated from malto-oligosaccharide substrates having 2-7 D-glucosyl residues indicated that subsites 1 and 2 had greater affinity for D-glucosyl residues in the Trp120----Tyr mutant than in wild-type glucoamylase. These results suggest that Trp120 from a distant subsite is crucial for the stabilization of the transition-state complex in subsites 1 and 2.

Stopped‐flow fluorescence and steady‐state kinetic studies of ligand‐binding reactions of glucoamylase from Aspergillus niger

“…lhus, E*L is much more stable than EL, and this is true for all ligands investigated. The binding energy of subsite 1 of this enzyme is positive, whereas that of subsite 2 is negative and numerically 10 times greatcr [9]. It seems impossible therefore, if the reaction proceeds as suggested for Rhizopus glucoamylase [lo], that binding at subsite 2, followed by relocation or sliding of the substrate to form a productive complex with the non-reducing sugar moiety at subsite 1, may take place (Fig.…”

“…4). shows an analysis of the experimental data according to Eqn (9). The resulting ~F,,, ([~],GC) and Kl are given in Table 1.…”

NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9–15‐ppm range

“…Trp residues replaced by Phe) were obtained from Novo Nordisk (Bagsvaerd, Denmark). The construction of the corresponding mutant genes and the expression of the cDNAs in Aspergillus has been published for the W120F mutant [27] and for others will be reported elsewhere (J. Lehmbeck, unpublished results). The mutant proteins were isolated by affinity chromatography on acarbose-Sepharose [28] followed by Q-Sepharose FPLC purification [29].…”

Molecular cloning of a glucoamylase gene from a thermophilic Clostridium and kinetics of the cloned enzyme

“…K,,, and k for isomaltose were 37-fold smaller and 22-fold larger than those of the Aspergillus niger enzyme [35], respectively, and 42-fold smaller and 6.4-fold larger than those of the Rhizopus delemar enzyme [35]. The binding energy at subsite 1 was negative, but almost all of the reported glucoamylases except for Hormoconis resinae glucoamylase P have positive or zero energy at subsite 1 [9,10,30,361 (Table 3).…”