# Mechanistic and Molecular Dynamics Studies Reveal that Increased Loop 3 Mobility Alters Substrate Capture in an NADH:Quinone Oxidoreductase

## TL;DR

This study shows how a flexible protein loop in an enzyme affects how substrates bind and react, using a combination of experiments and simulations.

## Contribution

The paper demonstrates that loop 3 mobility modulates substrate binding in NADH:quinone oxidoreductase through mechanistic and molecular dynamics studies.

## Key findings

- The P78G mutation increases loop 3 mobility, leading to a 3.5-fold decrease in K_CoQ0 and an 80-fold increase in K_d for NADH.
- Molecular dynamics simulations show that the P78G mutation allows loop 3 to sample more open conformations.
- More open gate conformations favor smaller substrate access but hinder interactions with bulkier substrates like NADH.

## Abstract

Dynamic protein loops
can act as molecular gates that stabilize
enzyme–substrate complexes, yet the underlying motions are
poorly defined. Here, we dissect the role of loop 3 in an NADH:quinone
oxidoreductase (NQO, UniProt Q9I4V0) from Pseudomonas
aeruginosa PA01 in governing substrate binding and
catalysis. Previous mechanistic and structural studies proposed that
loop 3 fluctuations regulate substrate binding; however, an associated
atomic-level understanding of the conformational changes is lacking.
We probe the role of loop 3 dynamics in substrate capture and catalysis
by mutating conserved P78 to glycine, which perturbs the gate rigidity.
Steady-state kinetics with NQO-P78G and NQO-WT at varying concentrations
of NADH and coenzyme Q0 established a 3.5-fold decrease
in the K
CoQ0 value, a 2.0-fold reduction
in the k
cat value, and a 1.8-fold increase
in the k
cat/K
CoQ0 value. The anaerobic reductive half-reaction of NQO-P78G with NADH
yielded a ≤3.5-fold decrease in the k
red value and an estimated 80-fold increase in the K
d value compared to NQO-WT. Molecular dynamics
simulations of ligand-free NQO-P78G and NQO-WT suggest that the P78G
mutation disrupts interdomain interactions, allowing loop 3 to sample
more open conformations. The combination of mechanistic and computational
experiments suggests that more open gate conformations minimally promote
access of the smaller coenzyme Q0 substrate to the active
site. In contrast, the bulkier NADH substrate is less likely to associate,
as the more open conformations prevent key interactions with NQO gate
residues from forming. These results build on previous studies with
NQO by demonstrating that altering loop 3 gate rigidity modulates
substrate binding.