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/KMvalues 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 Δ(ΔG) =-RT In [(Kcat/KM)deoxy/(Kcat/KM)hydroxy] for the series of analogues. The activity of all three enzymes was found to depend heavily on the 4-and 6-OH groups (4ʹ-and 6ʹ-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 (3ʹ-OH in maltose) was involved in weaker interactions, while the 2-OH (2ʹ-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/KMvalues obtained with substrate analogues for one enzyme is plotted against the corresponding values for a second enzyme. The high linear correlation coefficients (p) obtained, 0.916, 0.958, and 0.981, indicate significant similarity in transition-state interactions, although the three enzymes lack overall sequence homology. A short amino acid sequence, however, which is critical for glucoamylase activity (Clarke, A.J., & Svensson, B. (1984) Carlsberg Res. Commun. 49, 559-566; Sierks, M.R., Ford, C., Reilly, P.J., & Svensson, B. (1989) Protein Eng. 2, 621-625) was recognized in glucose oxidase. Whether this area also plays a crucial role in that enzyme is not yet known.
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