Frandsen T P, Stoffer B B, Palcic M M, Hof S, Svensson B
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
J Mol Biol. 1996 Oct 18;263(1):79-89. doi: 10.1006/jmbi.1996.0557.
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.
分子识别、定点诱变和分子建模相结合,以描述对黑曲霉葡萄糖淀粉酶 - 异麦芽糖复合物的形成和催化至关重要的氢键。对过渡态复合物能量学的分析确定,OH - 4'、- 6'和 - 4对异麦芽糖水解至关重要。与异麦芽糖OH - 4(还原端单元,即在葡萄糖淀粉酶结合亚位点2)形成氢键的侧链诱导底物构象调整,以优化由带电荷的氢键对非还原单元(即在亚位点1)的OH - 4'和 - 6'贡献的结合能。这些相互作用在葡萄糖淀粉酶和异麦芽糖的优选反式 - gauche构象的模拟复合物中很明显。动力学分析表明,异麦芽糖相应的脱氧和O - 甲基类似物的kcat降低了10³至10⁵倍。对亚位点2水平的两个突变体Glu 180→Gln和Asp 309→Glu的分析表明,OH - 3和 - 4对酶 - 过渡态复合物的结合能ΔΔG比野生型酶弱6 - 7 kJ mol⁻¹。然而,出乎意料的是,异麦芽糖OH - 4'和 - 6'(在亚位点1)的取代导致两个突变体的ΔΔG‡降低10至12 kJ mol⁻¹。因此,亚位点2的突变强烈干扰了远距离的过渡态稳定相互作用。这通过构象偏向的甲基6 - R - C - 甲基 - α - 异麦芽糖苷的4' - 和6' - 脱氧类似物得到证实,它们容易采用反式 - gauche构象,对两个突变体和野生型的ΔΔG‡均为18至20 kJ mol⁻¹。因此,在催化过程中,葡萄糖淀粉酶似乎诱导了从主要的溶液gauche - gauche构象异构体向反式 - gauche异麦芽糖的转变。这导致在酶过渡态复合物中亚位点1处的结合增强。