Stopped-flow fluorescence and steady-state kinetic studies of ligand-binding reactions of glucoamylase from Aspergillus niger.
The presteady-state and steady-state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition-state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial-rate, stopped-flow and steady-state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three-step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non-enzymic hydrolysis the transition-state stabilization energy of glucoamylase is approximately -66 kJ/mol with maltose and only -14 kJ/mol with isomaltose. Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.
Olsen
K
K
Chemical Laboratory IV, University of Copenhagen, Denmark.
Svensson
B
B
Christensen
U
U
D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism*; D005942:Gluconates / Q000378:metabolism; D007534:Isomaltose / Q000378:metabolism; D007700:Kinetics; D007783:Lactones; D008024:Ligands; D008320:Maltose / Q000378:metabolism; D008433:Mathematics; D008962:Models, Theoretical; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D005942:Gluconates; D007783:Lactones; D008024:Ligands; D007534:Isomaltose; D008320:Maltose; D005087:Glucan 1,4-alpha-Glucosidase; C010730:beta-glucono-1,5-lactone