# Structural mechanisms for cold‐adapted activity of phosphoenolpyruvate carboxykinase

## TL;DR

This paper explores how a cold-adapted enzyme maintains function at low temperatures by analyzing its structure and comparing it to a mesophilic counterpart.

## Contribution

The study reveals structural substitutions in a psychrophilic enzyme that hinder active site closure, providing a mechanistic basis for cold adaptation.

## Key findings

- Psychrophilic PEPCK has reduced catalytic efficiency and lower optimal temperature compared to mesophilic PEPCK.
- Structural substitutions in the Ω-loop increase the entropic cost of loop ordering and reduce enthalpic stabilization.
- These findings link specific structural features to altered kinetic behavior in cold adaptation.

## Abstract

Temperature is a critical factor in enzyme function, as most enzymes are thermally activated. Across Earth's diverse environments (−20 to 120°C), enzymes have evolved to function optimally at their organism's growth temperature. Thermophilic enzymes must resist denaturation, while psychrophilic enzymes must maintain activity with limited thermal energy. Although principles underlying thermostability are well established, the mechanisms governing kinetic adaptation to temperature remain less understood. To investigate this, we characterized the kinetics and determined a comprehensive series of X‐ray crystal structures of a psychrophilic, GTP‐dependent phosphoenolpyruvate carboxykinase (PEPCK) bound to substrates and non‐reactive mimics of the reaction coordinate. These structures were compared to those of a mesophilic PEPCK. PEPCK is a dynamic enzyme requiring substantial conformational changes during catalysis, particularly ordering of the active site Ω‐loop lid. The psychrophilic enzyme exhibited a reduced catalytic efficiency (k
cat/K
M) and lower optimal temperature (T
opt) relative to its mesophilic counterpart. Structural comparisons revealed substitutions in the Ω‐loop that likely increase the entropic cost of loop ordering and reduce enthalpic stabilization, hindering efficient active site closure. These results provide a mechanistic basis for cold adaptation in enzyme catalysis, linking specific structural features to altered kinetic behavior. Understanding such adaptations not only advances our knowledge of enzyme evolution but also informs protein engineering efforts aimed at designing efficient biocatalysts for industrial applications operating at non‐physiological temperatures.