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 + Gx (k1) reversible (k-1) EGX (k2)reversible(k-2) EP (kcat)--> 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 kcat value for wild-type GA with maltose, indicating a ronhydrolytic 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 alpha-D-glucosyl fluoride.
Natarajan
S K
SK
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County 21228, USA.
Sierks
M R
MR
D001230:Aspergillus / Q000201:enzymology*; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence; D014312:Trisaccharides / Q000378:metabolism
D014312:Trisaccharides; C008317:maltotriose; D008320:Maltose; D005087:Glucan 1,4-alpha-Glucosidase