The ability of MZP to inhibit a purified RNA capping enzyme has not been previously documented and has implications on our understanding of the catalytic mechanism of RNA capping enzyme. Our results indicate that the overall GTase reaction is inhibited by MZP. HCE is a bifunctional protein harboring both RTase and GTase activity. In the presence of MZP, the RTase activity appears to be relatively unaffected while the GTase activity is inhibited, indicating that the GTase activity is the main target of MZP inhibition. The GTase catalysis is a two-step reaction. Interestingly, the NVP-BEZ235 PI3K inhibitor inhibition of the complete GTase reaction by MZP could not be explained completely by the inhibition of its individual steps. While the GMP transfer onto an acceptor RNA does not contribute significantly to the general inhibition effect caused by MZP, the EpG formation is inhibited by MZP with an IC50 25-times higher than the complete GTase reaction. This weak inhibition, led us to hypothesize that two distinct MZP binding sites could be present on HCE. The main binding site responsible for the drug inhibitory potency is speculated to be allosteric and will be further discussed. The second would be the active site and is believed to bind MZP with a lower affinity, likely due to the drug chemical similarity with GMP. The weak binding of MZP to the active site would be coherent with EpG complex formation being competitively inhibited by MZP. In silico docking of MZP on the highly conserved active site of an open conformation homology model of HCE GTase domain provides additional information on the mechanism of MZP binding to this site. MZP appears to be coordinated by the most conserved amino acids, which would thereby explain the low inhibition specificity toward various GTase. Nevertheless, the mechanism of inhibition of the enzyme-GMP complex, which is mediated by the weak binding of MZP to the active site, is fundamentally different and weaker than the mechanism of the complete GTase inhibition. A complete GTase catalytic round does not only imply the formation of the EpG intermediate complex and the transfer of the GMP moiety to an acceptor RNA, but also involves complex conformational changes where the OB fold domain leans toward or away from the NT domain. MZP could bind HCE and block this conformational change, possibly through stabilization of the closed conformation, thereby preventing the reopening of the protein upon GTP hydrolysis. This hypothesis would likely imply an allosteric MZP binding site. Interestingly, the kinetics studies, although performed in steady-state condition, point toward a non-competitive mechanism of inhibition, which would indicate that MZP binds elsewhere than the active site. This is also coherent with the inability of MZP to be used as a substrate and transferred onto an acceptor RNA. Reversine 656820-32-5 Despite the very high degree of conservation among this specific family of nucleotidyltransferases, MZP harbors a 5-to 25-fold gain in specificity for HCE when compared to other GTases. This result additionally supports the presence of an allosteric MZP binding pocket. Oddly enough, the lone GTase domain of HCE is less susceptible by 10-fold to MZP inhibition than the full-length HCE. This evidence, not only supports the presence of an allosteric site, but can also provide additional information about its localization on HCE. It is tempting to speculate that MZP could bind near the N-terminal of the GTase domain or on a region of interaction.