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

Abstract

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)

Automated docking of glucosyl disaccharides in the glucoamylase active site

“…Similar binding was found at the first subsite for all a-linked substrates, with the same two critical hydroxyl groups, OH-4 A and OH-6 A , that had been identified by chemical mapping of maltose and isomaltose analogues 15,[26][27][28][29] serving as the anchors required for catalysis. At the second subsite, a critical unmodified hydroxyl group that promotes a strong interaction has been identified in each substrate.…”

“…Beside providing the stereochemical complementarity required for binding of carbohydrates to proteins, 24,25 key hydroxyl groups also establish essential polar interactions required for catalysis. Such groups have been identified in GA hydrolysis of maltose [26][27][28] and isomaltose 15,29 and also have been suggested for other a-linked glucopyranosyl disaccharides. 30 Conformational analysis of differently linked glucopyranosyl disaccharides has been conducted using MM3, [31][32][33][34] a force field whose low-energy conformations of glucose-containing disaccharides are in good agreement with the torsion angles of crystal structures.…”

“…23 A similar effect is expected for the remaining a-linked substrates, given the variations on k cat /K M found at the nonreducing-end monodeoxy analogues of maltose. 27 These hydroxyl groups are essential for catalysis, by taking approximately the same position in all cases in promoting interactions with the conserved residue Asp55 14,15,22,23 (Fig. 2).…”

Automated docking of monosaccharide substrates and analogues and methyl α-Acarviosinide in the glucoamylase active site

“…4f), in acarviosinide binding correlates with the importance of the same groups for effective maltose hydrolysis. 51,52,59 These hydrogen bonds are also found in the acarbose-GA complex. 9,11 The strong interaction with C-6 B correlates well with the predicted hydrophobic environment for the 6-position in the GA active site found in kinetic studies of 6-substituted maltose derivatives.…”

“…15,50 Both gt and tg conformations of the hydroxymethyl group are also possible and often lead to distinct clusters of docked structures, but they cause a molecular interaction energy penalty. The OH-6 A group is not only essential for hydrolysis of maltose and isomaltose, [50][51][52] but is equally necessary for the condensation of Dglucose and its deoxy derivatives. 3,53 The positional flexibility of this hydroxyl group in the GA active site could have some mechanistic implications both in substrate binding and in catalysis.…”