pH-dependence of the fast step of maltose hydrolysis catalysed by glucoamylase G1 from Aspergillus niger.
The presteady-state kinetic parameters of the interaction ofwild-type glucoamylase from Aspergillus niger (EC 3.2.1.3)with maltose were obtained and analysed in the pH range 3-7 withintervals of 0.25 pH units. In all cases the following three-step reaction scheme was found to apply. [Equation: see text]. The general result of the analysis of the presteady-state kinetics is that glucoamylase G1 is affected by the protonation states of three groups, with pK(a) values of 2.7, 4.5 and 5.7 in the free enzyme and of 2.7, 4.75 and 6.5 in the first enzyme-substrate complex. The protonation of the group in the enzyme-substrate complex with a pK(a) 6.5 hadno effect on k(2) (1640 s(-1)) or k(-2) (20+/-4 s(-1)), but resulted in a stronger enzyme-substrate interaction, due to a decrease of K(1) from 40 to 6.3 mM. In other words,when the substrate is bound, the pK(a) of the acidgroup changes to increase the fraction of reactive enzyme. Since this pK(a) parallels that of the Michaelis complex, known from the pH-dependence of k(cat), the group in question is most probably the catalytic acid Glu-179. Protonation of Glu-179 thus is of no importance in the second step, clearly indicating that this step represents a conformational change and not the actual hydrolysis step of the reaction. Protonation of the pK(a)=4.75 group leads to a small decrease in k(2) to 1090 s(-1), and also to minor changes in K(1). The group with pK(a)=2.7 leads toa major decrease of k(2), of which the limit may bezero, but shows no effect on K(1). Thus no differenceis seen between the pK(a) values of the free enzymeand of the first enzyme-substrate complex at low pH.
Christensen
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Kemisk Laboratorium IV, Universitetsparken 5, DK-2100 Copenhagen, Denmark.
D001234:Aspergillus niger / Q000201:enzymology* / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008320:Maltose / Q000378:metabolism*
D008320:Maltose; D005087:Glucan 1,4-alpha-Glucosidase