# Substrate cooperativity shapes competitive inhibitor responses in mycobacterial inosine 5′-monophosphate dehydrogenase

## TL;DR

This study explains how substrate cooperativity affects inhibitor responses in a key enzyme from mycobacteria, offering guidance for better drug testing.

## Contribution

The study reveals a pH-dependent cooperativity mechanism in mycobacterial IMPDH that influences inhibitor activity.

## Key findings

- IMPDH exhibits pH-dependent cooperativity through IMP-driven domain interactions in the tetramer.
- IMP-competitive inhibitors can paradoxically activate the enzyme under certain substrate conditions.
- Allosteric regulators like GTP and ppGpp show altered inhibition due to this cooperativity.

## Abstract

Inosine 5′-monophosphate dehydrogenase (IMPDH) is a promising antimicrobial target due to its central role in guanine nucleotide biosynthesis. Accurate and reliable kinetic measurements are essential for evaluating inhibitors. However, the enzyme’s complex reaction mechanism and substrate cooperativity complicate analysis, leading to inconsistent reports on IMPDH reaction kinetics in key pathogenic mycobacteria. Here, we present an in-depth biochemical analysis of mycobacterial IMPDH, revealing pH-dependent cooperativity mediated by IMP-driven interactions between catalytic domains within the tetramer. This mechanism may result in paradoxical activation by IMP-competitive inhibitors under specific substrate conditions. We further show that such effects may influence apparent inhibition by the natural allosteric regulators GTP and ppGpp. Based on these findings, we outline practical recommendations for designing kinetic experiments that reflect physiologic conditions with the aim of more accurately evaluating IMPDH inhibitors for drug discovery.