# Mechanistic Determinants of Oriented Enzyme Immobilization from Martini Simulations

## TL;DR

This study uses computer simulations to understand how enzyme immobilization affects enzyme structure and function, offering insights for designing better biocatalysts.

## Contribution

A new protocol using Go̅Martini simulations is introduced to explore enzyme immobilization effects on structure and function.

## Key findings

- Cluster-based immobilization restricts flexibility in surface-contacting subunits while preserving mobility in exposed regions.
- Surface attachment does not affect ethanol association rates but significantly slows NADH dissociation, reducing catalytic efficiency.
- Simulation results align with experimental findings, demonstrating the predictive power of the Go̅Martini model.

## Abstract

Although enzyme immobilization is widely used in biotechnology,
it still poses challenges as a result of the trade-offs among stability,
activity, and surface interactions. Computer simulations offer a promising
aid to exploring the effects of different immobilization sites and
surface chemistry on both the conformational dynamics and catalytic
activity of these biomolecules. Here, we introduce a protocol based
on a structure-based version of the Martini coarse-grained simulation
model (Go̅Martini) to explore how surface tethering geometry
influences the structure and function of immobilized Bacillus
stearothermophilus alcohol dehydrogenase (BsADH). We compare
traditional His-tag tethering with two engineered histidine cluster
variants, analyzing their behavior in both soluble and surface-tethered
states. We find that cluster-based immobilization locally restricts
flexibility in surface-contacting subunits while preserving the mobility
of exposed regions, resulting in an enhanced conformational stability
under thermal stress. Functional analyses reveal that the ethanol
association rates remain largely unaffected by surface attachment,
whereas the dissociation of NADH is significantly slowed, explaining
the reduced catalytic efficiency. These trends align with experimental
findings and highlight the predictive power of Go̅Martini simulations
in capturing key functional trade-offs. Altogether, this work offers
mechanistic insight into the rational design of immobilized biocatalysts
and outlines a practical framework for in silico exploration of enzyme–surface
systems.