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.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

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