| Abstrakt: |
Molecular recognition, site-directed mutagenesis, and molecular modeling are combined to describe hydrogen bonds important for formation and catalysis of theAspergillus nigerglucoamylase-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 preferredtrans-gaucheconformation. Kinetic analysis demonstrated reductions inkcatof 103to 105-fold for the corresponding deoxy- andO-methyl analogs of isomaltose. Analysis of two mutants at the level of subsite 2, Glu180 → Gln and Asp309 → Glu, showed the binding energy for the enzyme-transition state complex, ΔΔG, contributed by OH-3 and -4 to be 6 7 kJ mol−1weaker than with wild-type enzyme. Unexpectedly, however, substitution of isomaltose OH-4' and -6' (at subsite 1) resulted in 10 to 12 kJ mol−1lower ΔΔG 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-α-isomaltoside, readily adoptingtrans-gaucheconfor mation, that exhibit full ΔΔG of 18 to 20 kJ mol−1for both mutants and wild-type. Glucoamylase, during catalysis, thus seems to induce a change from the predominant solutiongauche-gaucheconformer totrans-gaucheisomaltose. This leads to enhanced binding at subsite 1 in the enzyme transition-state complex. |
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