Engineering Dynamic Surface Peptide Networks on Butyrylcholinesterase_G117H for Enhanced Organophosphosphorus Anticholinesterase Catalysis

Abstract

The single residue mutation of butyrylcholinesterase (BChEG117H) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278–285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in K M and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.

Allosteric communication in class A β-lactamases occurs via cooperative coupling of loop dynamics

“…Dynamical cross-correlation analysis provides information about the pathways of signal propagation and also some insights into the timescales of allosteric communication in TEM-1 and KPC-2 β-lactamases. Dynamic cross-correlation maps (DCCMs) have been previously used to identify networks of coupled residues in several enzymes ( Agarwal et al, 2004 ; Hester et al, 2019 ; Agarwal et al, 2012 ).…”

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

“…The dynamic cross-correlation maps (DCCM) have been previously used to identify networks of coupled residues in several enzymes by us. [75][76][77] Using a similar approach, Dynamic Cross-Correlation Maps (DCCMs) were calculated for the Apo EQ and IB EQ simulations and also for the Apo NE nonequilibrium simulations ( Figure 5). In these figures, the green regions represent no to slightly positive correlations, while yellow regions represent moderate negative correlations.…”

Enzyme Dynamics: Looking Beyond a Single Structure

“…New engineering solutions with photo‐activation of conformations and cross‐linking within dynamical residue networks have also been designed to improve enzyme catalysis and design de novo function . Further, loop engineering of dynamical surface residues to increase catalysis has also provided new means of validation …”