Energetic and mechanistic studies of glucoamylase using molecular recognition of maltose OH groups coupled with site-directed mutagenesis.
Molecular recognition using a series of deoxygenated maltose analogues was used to determine the substrate transition-state binding energy profiles of 10 single-residue mutants at the active site of glucoamylase from Aspergillus niger. The individual contribution of each substrate hydroxyl group to transition-state stabilization with the wild type and each mutant GA was determined from the relation Delta(DeltaG()) = -RT ln[(k(cat)/K(M))(x)/(k(cat)/K(M))(y)], where x represents either a mutant enzyme or substrate analogue and y the wild-type enzyme or parent substrate. The resulting binding energy profiles indicate that disrupting an active site hydrogen bond between enzyme and substrate, as identified in crystal structures, not only sharply reduces or eliminates the energy contributed from that particular hydrogen bond but also perturbs binding contributions from other substrate hydroxyl groups. Replacing the active site acidic groups, Asp55, Glu180, or Asp309, with the corresponding amides, and the neutral Trp178 with the basic Arg, all substantially reduced the binding energy contribution of the 4'- and 6'-OH groups of maltose at subsite -1, even though both Glu180 and Asp309 are localized at subsite 1. In contrast, the substitution, Asp176 --> Asn, located near subsites -1 and 1, did not substantially perturb any of the individual hydroxyl group binding energies. Similarly, the substitutions Tyr116 --> Ala, Ser119 --> Tyr, or Trp120 --> Phe also did not substantially alter the energy profiles even though Trp120 has a critical role in directing conformational changes necessary for activity. Since the mutations at Trp120 and Asp176 reduced k(cat) values by 50- and 12-fold, respectively, a large effect on k(cat) is not necessarily accompanied by changes in hydroxyl group binding energy contributions. Two substitutions, Asn182 --> Ala and Tyr306 --> Phe, had significant though small effects on interactions with 3- and 4'-OH, respectively. Binding interactions between the enzyme and the glucosyl group in subsite -1, particularly with the 4'- and 6'-OH groups, play an important role in substrate binding, while subsite 1 interactions may play a more important role in product release.
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore 21250, USA. sierks@umbc.edu
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D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004734:Energy Metabolism; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity; D013816:Thermodynamics
D008320:Maltose; D005087:Glucan 1,4-alpha-Glucosidase