The tertiary structure of proteins has been represented as a network, in which residues are nodes and their contacts are edges. Protein structure networks contain residues, called hubs or central, which are essential to form short connection pathways between any pair of nodes. Hence hub residues may effectively spread structural perturbations through the protein. To test whether modifications nearby to hub residues could affect the enzyme active site, mutations were introduced in the β-glycosidase Sfβgly (PDB-ID: 5CG0) directed to residues that form an α-helix (260-265) and a β-strand (335-337) close to one of its main hub residues, F251, which is approximately 14 Å from the Sfβgly active site. Replacement of residues A263 and A264, which side-chains project from the α-helix towards F251, decreased the rate of substrate hydrolysis. Mutation A263F was shown to weaken noncovalent interactions involved in transition state stabilization within the Sfβgly active site. Mutations placed on the opposite side of the same α-helix did not show these effects. Consistently, replacement of V336, which side-chain protrudes from a β-strand face towards F251, inactivated Sfβgly. Next to V336, mutation S337F also caused a decrease in noncovalent interactions involved in transition state stabilization. Therefore, we suggest that mutations A263F, A264F, V336F and S337F may directly perturb the position of the hub F251, which could propagate these perturbations into the Sfβgly active site through short connection pathways along the protein network.
Submitter: Shu-Ching Ou
Submission Date: March 28, 2019, 2:17 p.m.
|Number of data points||303|
|Assays/Quantities/Protocols||Experimental Assay: kcat/Km ; Experimental Assay: kcat ; Experimental Assay: Km ; Derived Quantity: SD of ΔΔG‡ ; Derived Quantity: ΔΔG‡ ; Derived Quantity: SD of kcat/Km ; Derived Quantity: SD of kcat ; Derived Quantity: SD of Km|
|Libraries||Variants for beta-glycosidase_substrate|