Investigating the Consequences of Non-active site Mutations on the Structure, Function and Dynamics of the Molten Globule Enzyme Monomeric Chorismate Mutase
Intrinsically disordered enzymes serve as useful models to understand their catalytic function against the backdrop of an unstructured protein. The characteristic flexibility in conformation seen in IDPs is a rare occurrence among enzymes and one such enzyme is the engineered protein: monomeric Chorismate Mutase (mCM). mCM surprisingly retains similar enzyme activity as its parent dimeric protein Chorismate Mutase from Methanococcus jannaschii (MjCM) despite losing the ordered globular structure. In this work using a previously demonstrated transition state analogue (TSA), we analyze the structural transitions in mCM during catalysis. Additionally, consequences of three non-active site single point mutations were investigated using CD; Trp-Dansyl FRET measurements using fluorescence lifetime; and time-resolved fluorescence anisotropy measurements; to map the local (near Trp) and global structural transitions in mCM during catalysis. Mutant2 (W24K + C69A); and Mutant3 (W24K + C69A + A6C); revealed a 97 and 89% drop-in activity compared to mCM; quite unlike Mutant1 (W24K, 19% drop). Mutant1 as opposed to Mutant3 was most sensitive to binding of TSA as quantified by structural displacement measured using FRET. This was consistent with an overall globular structure compaction induced by TSA binding in Mutant1 as reflected by a dip in rotational correlation time of Cys-conjugated dansyl probe from 10.3 to 8.4 ns. Our results highlight the critical role of Cys69 residue, that is ~19 [A] away from mCM active site, in influencing the hydrophobic collapse upon substrate binding and subsequent catalytic activity.