My graduate research has focused on the enzymology of protein S-palmitoylation, a reversible posttranslational modification catalyzed by DHHC palmitoyltransferases. When I started my thesis work, the structure of DHHC proteins was not known. I sought to purify and crystallize a DHHC protein, identifying DHHC20 as the best target. While working on this project, I came across a protein of unknown function called metallo-β-lactamase domain-containing protein 2 (MBLAC2). A proteomic screen utilizing affinity capture mass spectrometry suggested an interaction between MBLAC2 (bait) and DHHC20 (hit) in HEK-293 cells. This finding interested me initially from the perspective of finding an interactor that could help stabilize DHHC20 into forming better quality crystals as well as discovering a novel protein substrate for DHHC20. I was intrigued by MBLAC2 upon learning that this protein is predicted to be palmitoylated by multiple proteomic screens. Additionally, sequence analysis predicts MBLAC2 to have thioesterase activity. Taken together, studying a potential new thioesterase that is itself palmitoylated was deemed to be a worthwhile project. When the structure of DHHC20 was published in 2017, I decided to switch my efforts to characterizing MBLAC2. This dissertation consists of three chapters. Chapter 1 is an introductory chapter surveying the literature on protein S-fatty acylation, with an emphasis on the mechanistic and functional studies on palmitoylation and DHHC proteins. The review also touches on the current status and advancements in the relatively understudied but equally important field of protein depalmitoylation. Chapter 2 outlines my efforts to crystallize the palmitoyltransferase DHHC20 with the goal of solving its 3D structure. Additionally, I present the results of my investigation of the oligomerization of DHHC20 at a single molecule level. Chapter 3 describes my biochemical study of the uncharacterized MBLAC2 protein. I discuss my findings that MBLAC2 is a palmitoylated protein and is a substrate of DHHC20. My subsequent functional studies reveal that MBLAC2 is a robust acyl CoA thioesterase and contains multiple zinc-binding residues that are important for catalytic activity. Lastly, a book chapter we published in Methods of Molecular Biology on purifying recombinant DHHC proteins is included as an appendix.