Abstract:
Understanding all parameters contributing to enzyme activity is crucial in enzyme catalysis. For enzymatic PET degradation, this involves examining the formation of the enzyme-PET complex. In IsPETase (WT), a PET-degrading enzyme from Ideonellasakaiensis, mutating two non-catalytic residues (DM) significantly enhances activity. Such mutations, depending on their position in the tertiary structure, fine-tune enzyme function. However, detailed molecular insights into these mutations\' structurefunction relationship for PET degradation are lacking. This study characterizes IsPETase\'s catalytic ability compared to WT TfCut2 using molecular dynamics simulations and quantum mechanical methods. We explore the conformational landscape of the enzyme-PET complex and quantify residue-wise interaction energy. Notably, aromatic and hydrophobic residues Tyr, Trp, and Ile in the catalytic subsite S1, and aromatic Phe and polar Asn in the anchoring subsite S3, crucially optimize PET binding. These residues enhance PET specificity over non-aromatic plastics. Our findings suggest that the balance between binding at subsite S1 and subsite S3, which is influenced by cooperative mutations, underlies catalytic activity. This balance shows a positive correlation with experimentally obtained kcat/Km values: WT TfCut2 < WT IsPETase << DM IsPETase.
摘要:
了解有助于酶活性的所有参数在酶催化中是至关重要的。对于酶促PET降解,这涉及检查酶-PET复合物的形成。在IsPETase(WT)中,来自Ideonellasakaiensis的PET降解酶,突变两个非催化残基(DM)显著增强活性。这样的突变,根据它们在三级结构中的位置,微调酶的功能。然而,缺乏对PET降解这些突变的结构功能关系的详细分子见解。本研究使用分子动力学模拟和量子力学方法描述了IsPETase与WTTfCut2相比的催化能力。我们探索了酶-PET复合物的构象景观,并量化了残基间的相互作用能。值得注意的是,芳香和疏水残基Tyr,Trp,和催化亚位点S1中的Ile,以及锚定亚位点S3中的芳族Phe和极性Asn,至关重要地优化了PET结合。这些残留物提高了PET的特异性,超过了非芳香族塑料。我们的发现表明,S1和S3亚位点的结合之间的平衡受到协同突变的影响,是催化活性的基础。该平衡显示与实验获得的kcat/Km值呈正相关:WTTfCut2