# Inactive but Essential: The Role of the Inactive State of E49 in the Mechanism of the Alpha Subunit of Tryptophan Synthase and Its Stand-Alone Blueprint ZmBX1

## TL;DR

This paper explains how the inactive state of a key amino acid in an enzyme helps improve its catalytic efficiency.

## Contribution

The study reveals how inactive conformations of E49 enhance catalytic efficiency in ZmBX1 through combined MD and DFT analysis.

## Key findings

- ZmBX1 has a catalytic efficiency 144,000 times higher than ZmTrpA due to E49's dual active/inactive states.
- Inactive states of E49 prevent reverse reactions after product formation, improving catalytic efficiency.
- Conformational changes and chemical steps are interlinked in the catalytic mechanism of ZmBX1.

## Abstract

The stand-alone version
of the alpha subunit of tryptophan synthase
(TrpA), ZmBX1, catalyzes the retro-aldol cleavage
of indole-3-glycerol phosphate (IGP) at a catalytic efficiency that
is approximately 144,000 times higher than that of isolated ZmTrpA. Available X-ray crystal structures of ZmBX1 and several TrpAs revealed identical overall structures as well
as active site geometries, showing high flexibility of the catalytic
E49 in both cases. Based on the crystallographic data, E49 was found
to adopt an active state in which the carboxylate group is close to
IGP for promoting the retro-aldol cleavage as well as an additional
inactive state whose catalytic function was unclear. In this work,
by using a combination of Molecular Dynamics (MD) simulations and
cluster model DFT calculations, we rationalize the effect of the active/inactive
conformation of the catalytic E49, as well as how L2 containing the
other catalytically relevant residue D60 affects catalysis. The higher
levels of retro-aldol activity observed for ZmBX1
are attributed to its dual ability to adopt not only active states
of the catalytic E49 crucial for retro-aldol cleavage but also inactive
states that position E49 in a noncatalytic orientation for disfavoring
the reverse aldol reaction back to IGP after product formation. Our
combined MD and QM studies elucidate the mechanistic interplay between
conformational changes and catalytic steps in ZmBX1
and TrpA enzymes. This study highlights the importance of optimizing
the conformational changes and chemical steps along the catalytic
itinerary for altering and/or improving enzymatic function.