To establish our FabH enzyme is active we performed HPLC assays with acetyl-CoA and malonyl-CoA, Figure . We chose malonyl-CoA rather than malonyl-S-ACP for a few reasons. First to avoid the need for coupled enzyme assays with FabD to generate malonyl-S-ACP, since FabD is also expected to be inhibited by the malonyl-CoA analogs. In addition we needed to avoid generating ACPs with substrate analogs attached for use in enzyme inhibition assays. Another reason is that malonyl-CoA has been used as a FabH substrate in drug screening platforms or in other studies with FabH homologs, but is incompletely characterized as a substrate. One complication of our HPLC assay is that the substrate acetyl-CoA peak overlaps with the product, acetoacetyl-CoA, although the acetoacetyl-CoA peak is distinctly broader, Figure . Nevertheless, the disappearance of malonyl-CoA and appearance of CoA is easily monitored to provide kinetics, Figure and. In these HPLC assays with 10 μM FabH and equimolar acetyl-CoA and malonyl-CoA at ~125 or ~250 µM at pH 8, the initial rates are 0.58±0.01 and 0.81±0.01 min -1 respectively, suggesting FabH is still not saturated even at these high concentrations. We also found FabH activity increases modestly with pH, Supplemental Figure and Table . The progress curves of B), F), and N) were fit with exponential decay or increase, while J), O), and P) were fit with a hysteretic/inhibition model as described in the supplemental information. The HPLC data points in the kinetic analysis are shown as green circles for CoA, red squares for malonyl-CoA and yellow or black diamonds for acetyl-or acetoacetyl-CoA. Notice the significant lag phase in J), O) and P). In order to determine the reactivity or inhibition behavior of FabH with acetyl-and malonyl-CoA analogs, we first explored their stability in the presence of FabH, see Figure panels G, H, K and L. Our synthesis of the standards oxa and aza(dethia)CoA, allowed us to confirm the presence or absence of the hydrolysis products and acetyl-oxa/aza(dethia)CoA allowed us to confirm decarboxylation products. As a baseline, we determined the stability of acetyl-and malonyl-CoA at pH 6, 7 and 8, Figure and Supplemental Figure . Non-enzymatic hydrolysis of acetyl-and malonyl-CoA is very slow at pH 6 and 7 with rates of ~0.0004 min -1 , while at pH 8 the rate is ~10 times higher. The analogs are completely stable in buffer at pH 6, 7 and 8 over 24 hours. The hydrolysis of acetyl-CoA by FabH in the absence of malonyl-CoA is insignificant compared to the non-enzymatic rates of hydrolysis, while the oxa(dethia) analogs hydrolyze very slowly and the aza(dethia) analogs are completely stable over 24 hours, Figure -H/K/L and Table .