In order to obtain inhibition constants, we used a continuous UV-Vis assay for the production of acetoacetyl-CoA. Previously a discontinuous UV-Vis assay based on 5,5′-dithiobis(2nitrobenzoic acid) (DTNB, Ellman's reagent) as a probe of CoA production was used to examine the inhibition of FabH by small molecules. However, we found that incubation of FabH with DTNB resulted in some precipitation of the protein and we were concerned that the CoA-TNB adduct could inhibit FabH. Rather, we chose to follow the production of acetoacetyl-CoA, which has a reasonable extinction coefficient at 302 nm of 17.7 mM -1 cm -1 in the presence of 50 mM Mg 2+ at pH 8.0 (see Supplemental Figure for extinction coefficient determination under assay conditions) and has been previously described for FabH at pH 7 where the extinction coefficient is lower. Comparison of FabH enzymatic reactions in the presence and absence of Mg 2+ followed by HPLC revealed no inhibition by 50 mM Mg 2+ . Using the UV-Vis assay, we determined an apparent Km of 0.3±0.1 µM for acetyl-CoA with 125 µM malonyl-CoA, Figure . With acetyl-CoA at 125 µM and varying malonyl-CoA, we noticed substrate inhibition at high malonyl-CoA concentrations. Fitting the data to a substrate inhibition model gave a malonyl-CoA Km of 2.5±0.8 mM, a Ki of 1.9±0.6 mM, a kcat of 5.9±1.4 min -1 and kcat/Km of 0.0024 µM -1 •min -1 , Figure . In previous reports at pH 7.0 with malonyl-S-ACP as an acceptor, the observed kcats are on the order of 2-3 s -1 , with an apparent KM of 5 µM for malonyl-ACP and 40 µM for acetyl-CoA, approximate kcat/KM of 24 µM -1 •min -1 and 3 µM -1 •min -1 respectively. Previous experiments using malonyl-CoA as a substrate at 5 mM but pH 7 determined a Vi of ~3 min -1 , which is close to our result of ~6 min -1 at pH 8. Comparison of kcat/KM demonstrates malonyl-S-ACP is a better substrate than malo-nyl-CoA by four orders of magnitude. To determine the importance of the malonyl-CoA nucleotide, we explored FabH catalysis with malonyl-S-pantetheine, see supplemental information for synthesis. Surprisingly, malonyl-S-pantetheine is an even poorer substrate than malonyl-CoA with a kcat/KM that is 20 times lower, further demonstrating the importance of the ACP for FabH catalysis. As such, we did not attempt to determine inhibition constants for the acetyl-and malonyl-pantetheine substrate analogs. We expect follow up studies comparing structures of FabH in the presence of malonyl-CoA and malonyl-S-ACP analogs will reveal how the significant substrate specificity is determined, but these studies need to be guided by knowledge of analog behavior with the enzyme.