Palmitoylation refers to the post-translational attachment of a 16-carbon fatty acid to proteins via a thioester bond at cysteine residues. To date, several hundred palmitoyl proteins have been identified in cells, including many proteins active in the pathogenesis of disease. DHHC palmitoyltransferases are integral membrane proteins that catalyze the addition of palmitate to intracellular substrate proteins. Little is known about the structure of DHHC proteins and how conserved elements contribute to the catalytic mechanism of these enzymes. No crystal structure of a DHHC protein has been reported, and the sequence of the DHHC protein catalytic domain contains no predicted secondary structural elements. This work focuses on understanding the palmitoylation, structure, and function of the catalytic cysteine rich domain (DHHCCRD) of DHHC3. DHHC proteins employ a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate and subsequently transfers that palmitate to a substrate protein. Mutation of the cysteine in the DHHC motif to serine (DHHS) blocks autoacylation and transfer activity in vitro, suggesting that the DHHC cysteine is the lone site of palmitoylation in the autoacylated intermediate. I developed a custom mass spectrometry method that directly identified two novel palmitoylation sites in the iii catalytic DHHC-CRD of DHHC3. A subsequent indirect mass spectrometry analysis identified additional palmitoylation sites in this domain. Mutation of identified palmitoylation sites and other conserved cysteines in DHHC3 dramatically reduced enzyme activity and altered the tertiary structure of the DHHC-CRD. These biochemical characteristics were shown to be the result of destabilized zinc binding in the DHHC-CRD. The removal of palmitate resulted in no dose dependent impact on the activity or structure of DHHC3 as revealed by limited proteolysis assays. As the cysteines mutated in this study are highly conserved, and have been associated with similar biochemical characteristics when mutated in other DHHC proteins, I propose that zinc binding is a conserved structural feature of the catalytic domain of DHHC proteins. iv