We found that FabH generates acetoacetyl-CoA via decarboxylation of malonyl-CoA to acetyl-CoA and reaction with a second malonyl-CoA at pH 7 and 8, but not pH 6, Figure /J and Supplemental Figure . The progress curves display a significant lag phase that we modeled as hysteresis alleviated by acetyl-CoA as outlined in the supplemental information and Supplemental Figure . We noticed that the lag phase was dependent on the stock of malonyl-CoA used to initiate the reaction and concentration. All commercial stocks of malonyl-CoA we tested had ~5-10% acetyl-CoA and ~5-10% CoA contaminants. To determine the behavior of FabH in the absence of acetyl-CoA with malonyl-CoA alone, we synthesized and purified an appropriate stock of malonyl-CoA as described in the supplemental information. FabH behavior with the acetyl-CoA free malonyl-CoA stock has a significant lag phase, Figure /J and Supplemental Figure . The addition of 10% acetyl-CoA alleviates the lag phase completely with the introduction of a burst phase that we interpret as the rapid use of acetyl-CoA, Figure . The second phase allowed us to determine the rate of malonyl-CoA conversion to acetoacetyl-CoA by activated FabH which is about 20 times slower than the complete reaction, Table . We interpret the lag phase to be FabH in an inactive state that only slowly decarboxylates malonyl-CoA, but acetyl-CoA binding generates a catalytically efficient enzyme that only slowly relaxes back to the inactive state, a similar state transition has been discussed previously. The finding that acetyl-CoA activates FabH for decarboxylation, even upon being expended from the reaction, inspired us to determine the activating potential of 1 and 2. Incubation of FabH with 125 µM of 1 or 2 then addition of 250 µM malonyl-CoA still yields progress curves with lag phases for the production of acetoacetyl-CoA, revealing 1 or 2 are not strong activators, Figure /P. The data was fit similarly to the progress curves of malonyl-CoA alone, but with inhibition as shown in the supplemental information. Fitting suggested both 1 and 2 act as inhibitors since the time to completion is longer (compare Figure with 2O or 2P), however more data is needed to generate reliable inhibition values. Rather than HPLC analysis, we describe a UV-Vis assay to assess inhibition below. FabH C112Q was ¼ the rate of malonyl-CoA decarboxylative Claisen condensation by the wild-type enzyme at pH 8. The lack of an electrophile or appropriate proton donor in the active site is expected for FabH C112Q, thus water is likely to fulfill that role, accounting for the lower rate of decarboxylation. FabH C112Q decarboxylation of malonyl-CoA is 5 times faster than activated wild-type in the absence of acetyl-CoA and there is no lag phase, suggesting FabH C112Q represents an activated acyl-enzyme state. The stability of the analogs against decarboxylation by FabH C112Q again reveals they will be useful for structure-function studies.