Identification of enzyme-substrate and enzyme-product complexes in the catalytic mechanism of glucoamylase from Aspergillus awamori

Intermediates in the catalytic mechanism of Aspergillus awamori glucoamylase (GA) were identified by studying pre-steady-state and steady- state kinetics of the wild-type GA/maltose and Trp120 → Phe GA/maltotriose reactions in H₂O and D₂O. Pre-steady-state fluorescence signal analysis was carried out to ascertain the relative intrinsic fluorescence of the enzyme intermediates. A three-step minimal pathway for oligosaccharide hydrolysis represented by E + G(x) (k₁) ⇆ (k_-1) EG(x) (k₂) ⇆ (k_-2) EP (k(cat)) → E + P is proposed. The first step, represented by the association constant K₁ (k₁/k_-1), depicts the fast formation of enzyme-substrate complex and is the primary factor in fluorescence quenching. A 2.7-fold increase in K₁ with D₂O as solvent is observed with both enzymes due to the cumulative effect of deuterium on complex hydrogen bonding at the active site. The second step further quenches the enzyme fluorescence and is identified as the hydrolytic step, forming an enzyme-product complex. Both k₂ and k_-2 values show similar 2-fold decreases in D₂O for both enzymes, consistent with the microscopic reversibility of the hydrolytic reaction. The solvent isotopic effect on the hydrolytic step is likely due to either abstraction of an exchangeable proton from the general acid Glu179 or directed addition of water to the oxocarbonium ion intermediate by the general base Glu400. No significant isotope effect was observed on the steady-state k(cat) value for wild-type GA with maltose, indicating a nonhydrolytic step as rate-limiting. The third step, a posthydrolytic rate-determining step, is the product release as evident from steady-state kinetics with wild-type and Trp120 → Phe GAs using α-D-glucosyl fluoride.