Thioesters are highly reactive centers for acyl-CoAs which allows them to be utilized in a variety of differing enzyme chemistries. As a result of this reactivity, structure-function studies of enzymes using acyl-CoA substrates is difficult. When acyl-CoAs are used in structure-function studies, they often result in a hydrolyzed CoA substrate fragment bound in the active site or require only one of multiple substrates in order to be bound. This results in a lack of information regarding enzyme interactions with the key thioester and acyl chain. To overcome this challenging problem, I have synthesized acetyl- and malonyl-CoA analogs where the thioester has been replaced by an ester (oxygen), amide (nitrogen), or carbonyl (carbon) in a way that is easier, cheaper, and more efficient than performed previously. In addition, we used our synthetic analogs to study a enzymes which span different acyltransferase mechanisms in a combination of kinetics and structure. With this work, it was determined that the amide analogs were stable in all enzymes it was utilized for, while the ester analogs were mostly stable, except the acetyl analog in KasIII, where it acted as a pseudo substrate. As such, these synthetic analogs may have future potential in either type of enzyme for structure-function studies, albeit limited for the acetyl ester analog.