Acetyl-CoA synthetase catalyzes the activation of acetate by the acetylation of the thiol group of Coenzyme A, while hydrolyzing ATP to AMP and pyrophosphate. The Arabidopsis thaliana acetyl-CoA synthetase (atACS) was compared to other acyl-CoA synthetases, and was computationally modeled on the available crystal structures of the Saccharomyces cerevisiae ACS1 and Salmonella enterica ACS. This allowed the identification of the residues that make up the putative carboxylate binding pocket residues. To further understand substrate selectivity and binding within the putative carboxylate binding pocket, selected residues were mutated to resemble the homologous residues in the Pseudomonas chlororaphis isobutyryl-CoA synthetase. Four residues (Ile323, Thr324, Val399, and Trp427) were identified that are proposed to form the carboxylate binding pocket. One residue, Trp427 was found to be the primary residue in determining the chain length of acceptable carboxylate substrates. By combing two mutations (Val399Ala, and Trp427Gly) the enzyme was able to utilize butyrate with a catalytic efficiency similar to the wild-type enzyme with acetate. Circular dichroism (CD) was used to evaluate the secondary structure of the wild-type atACS and the mutated variants. The CD spectra showed no difference between the mutated variants and the wild-type and indicated the enzyme is largely composed of α-helices.