# Catalytic pK a Attenuation in a Hydrolytic Metalloenzyme by Genetic Code Expansion

## TL;DR

Scientists used a new method to modify an enzyme's metal-binding site, making it work better in acidic conditions.

## Contribution

Genetic code expansion was used to alter a metalloenzyme's coordination sphere, improving its performance at lower pH.

## Key findings

- Substituting a histidine residue with Nπ-methyl-l-histidine improved the enzyme's tolerance to acidic conditions.
- The modification led to a decrease in the enzyme's catalytic pK a and changes in rate constants.
- The approach demonstrated the effectiveness of non-canonical amino acid-based engineering in enzyme optimization.

## Abstract

Hydrolytic metalloenzymes employ Lewis-acidic metal cofactors
to
activate water molecules, generating nucleophilic hydroxide species
that facilitate catalysis. Their catalytic efficiency across a wide
pH range is often governed by the protonation state of the metal-bound
water, reflected in pK
a values typically
between 6.8 and 9. Modulating this parameter is key to expanding enzymatic
activity for improved activity at neutral to acidic pH. Herein, we
apply genetic code expansion to mutate the primary metal-coordination
sphere of a model metallohydrolase: the dizinc phosphotriesterase
from Pseudomonas diminuta. Substitution
of the most catalytically indispensable coordinating histidine residue
(H55) to N
π-methyl-l-histidine
(πMH) resulted in substantial enzyme yields, efficient metal
coordination for either Zn2+ or Co2+, and up
to 5-fold improved tolerance to acidic conditions. Detailed mechanistic
analysis revealed a systematic decrease in catalytic pK
a and attenuation of several catalytic rate constants.
These results add to the growing body of evidence demonstrating the
power of ncAA-based engineering for refined tuning of enzyme properties.