Structure and energetics of the glucoamylase-isomaltose transition-state complex probed by using modeling and deoxygenated substrates coupled with site-directed mutagenesis.
Molecular recognition, site-directed mutagenesis, and molecular modeling are combined to describe hydrogen bonds important for formation and catalysis of the Aspergillus niger glucomylase-isomaltose complex. This analysis of the energetics of the transition-state complex identified OH-4', -6', and -4 as critical for isomaltose hydrolysis. Side-chains hydrogen bonded to isomaltose OH-4 (reducing end unit, i.e. at glucoamylase binding subsite 2) induced substrate conformation adjustment to optimize binding energy contributed by charged hydrogen bonds to OH-4' and -6' at the non-reducing unit (i.e. at subsite 1). These interactions were evident in the modeled complex of glucoamylase and isomaltose in the preferred trans-gauche conformation. Kinetic analysis demonstrated reductions in kcat of 10(3) to 10(5)-fold for the corresponding deoxy- and O-methyl analogs of isomaltose. Analysis of two mutants at the level of subsite 2, Glu 180-->Gln and Asp 309-->Glu, showed the binding energy for the enzyme-transition state complex, delta delta G, contributed by OH-3 and -4 to be 6-7 kJ mol-1 weaker than with wild-type enzyme. Unexpectedly, however, substitution of isomaltose OH-4' and -6' (at subsite 1) resulted in 10 to 12 kJ mol-1 lower delta delta G++ for both the mutants. Mutation at subsite 2, therefore, strongly perturbed distant transition-state stabilizing interactions. This was confirmed with 4'- and 6'-deoxy analogs of the conformationally biased methyl 6-R-C-methyl-alpha-isomaltoside, readily adopting trans-gauche conformation, that exhibit full delta delta G++ 18 to 20 kJ mol-1 for both mutants and wild-typ-. Glucoamylase, during catalysis, thus seems to induce a change from the predominant solution gauche-gauche conformer to trans-gauche isomaltose. This leads to enhanced binding at subsite 1 in the enzyme transition-state complex.
Frandsen
T P
TP
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Stoffer
B B
BB
Palcic
M M
MM
Hof
S
S
Svensson
B
B
D001234:Aspergillus niger / Q000201:enzymology*; D002240:Carbohydrate Sequence; D004187:Disaccharides / Q000737:chemistry; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007534:Isomaltose / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry; D011487:Protein Conformation
D004187:Disaccharides; D008024:Ligands; D009844:Oligosaccharides; C091668:methylisomaltoside; D007534:Isomaltose; D005087:Glucan 1,4-alpha-Glucosidase