Active site similarities of glucose dehydrogenase, glucose oxidase, and glucoamylase probed by deoxygenated substrates.

The specificity constants, kcat/KM, were determined for glucose oxidase and glucose dehydrogenase using deoxy-D-glucose derivatives and for glucoamylase using deoxy-D-maltose derivatives as substrates. Transition-state interactions between the substrate intermediates and the enzymes were characterized by the observed kcat/Km values and found to be very similar. The binding energy contributions of individual sugar hydroxyl groups in the enzyme/substrate complexes were calculated using the relationship delta(delta G) = -RT ln [(kcat/KM)deoxy/(kcat/KM)hydroxyl] for the series of analogues. The activity of all three enzymes was found to depend heavily on the 4- and 6-OH groups (4'- and 6'-OH in maltose), where changes in binding energies from 10 to 18 kJ/mol suggested strong hydrogen bonds between the enzymes and these substrate OH groups. The 3-OH (3'-OH in maltose) was involved in weaker interactions, while the 2-OH (2'-OH in maltose) had a very small if any role in transition-state binding. The three enzyme-substrate transition-state interactions were compared using linear free energy relationships (Withers, S. G., & Rupitz, K. (1990) Biochemistry 29, 6405-6409) in which the set of kcat/KM values obtained with substrate analogues for one enzyme is plotted against the corresponding values for a second enzyme. The high linear correlation coefficients (rho) obtained, 0.916, 0.958, and 0.981, indicate significant similarity in transition-state interactions, although the three enzymes lack overall sequence homology.(ABSTRACT TRUNCATED AT 250 WORDS)