Theoretical Studies Of The Catalytic Mechanism Of The Glutamate Decarboxylase And Adenosine2503Methyltransferase

In this thesis, we studied the catalysis mechanism of two types of enzymes using theoretical and computational chemistry, the glutamic acid decarboxylase (GAD) that uses pyridoxal5’-phosphate (PLP) as cofactor, and the methyltransferase that uses S-adenosylmethionine (SAM) as the cofactor.PLP-dependent Glutamic Acid DecarboxylaseThe production of gamma-aminobutyric acid (GABA) is catalyzed by two isoforms of GAD, using PLP as the cofactor. Between the two enzymes, GAD67accounts for normal GABA requirement, while GAD65stays inactive until emergent demand for GABA. Recent crystal structure findings revealed that the distinct conformation of a common catalytic loop of the enzymes may explain their different roles. In this paper, we studied the underlying reaction mechanism of the two isoforms of glutamic acid decarboxylase using density functional theory (DFT) with B3LYP hybrid functional. An almost complete reaction pathway with two branches in the late stage is identified for a fairly large model enzyme system, including9transition state (TS) structures and14intermediate (IM) structures. By comparing the transition barriers of two different reaction branches, we offer an explanation for the distinct functions of the two GAD isoforms. The results on one hand helps to enrich our understanding of the working principles of GAD and other PLP-assisted enzymes, while on the other hand shed light on the rational design of transition state analog inhibitors for glutamate decarboxylase.SAM-Dependent MethyltransferaseMethylation of ribosome is important for translational fidelity, correct ribosomal assembly and bacterial antibiotic resistance. Radical S-Adenosylmethionine (SAM) enzymes catalyze most of the processes. It has long been believed that two S-Adenosylmethionine (SAM) molecules serve as the cofactor by providing the5-deoxyadenosyl radical (5-dA·) for activating the substrate for methylation, as well as the methyl. Recently, an alternative mechanism has been found by Booker et al.(Science332,604(2011)) for a pair of radical SAM enzymes, RlmN and Cfr, which respectively methylate the C2and C8positions of adenosine2503on23S RNA. The deuterium isotope labeling experiments reveal that the methyl group does not transfer directly from SAM to the adenosine, as previously believed. Instead it passes to Cys355first, then onto adenosine. In this paper, we study the proposed reaction mechanism using density functional theory (DFT) with B3LYP hybrid functional. We identified the structures of the transition states and the intermediates, and calculated their free energies. The activation barrier results indicate that the proposed reaction mechanism is plausible. The rate-limiting step is found to be the formation ofa disulfide bond.