Structure, Mechanism and Regulation of human UMP Synthase
by Laura Liliana Kirck
Date of Examination:2024-11-25
Date of issue:2025-01-10
Advisor:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Ricardo A. Mata
Persistent Address:
http://dx.doi.org/10.53846/goediss-11000
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Abstract
English
Enzymes are of critical importance for a large range of biochemical reactions, catalyzing the conversion of a substrate to a product by lowering the energy of the transition state. The resulting rate acceleration in comparison to the uncatalyzed reaction can be enormous. A prominent example is the 10^17-fold enhancement of the decarboxylation of orotidine 5’-monophosphate (OMP) to uridine 5’-monophosphate (UMP), making the responsible enzyme, orotidine 5’-monophosphate decarboxylase (OMPDCase), one of the most efficient enzymes known. However, the mechanism behind this improvement is still unclear and widely discussed to this date. Furthermore, possible rate-limiting bottlenecks have remained unresolved. This thesis provides a detailed analysis of the active site residues of human OMPDCase. In order to study the reaction mechanism, the residues of the catalytic center were substituted with either chemically or structurally similar, or completely dysfunctional amino acids via site-directed mutagenesis. The results of the 23 analyzed variants elucidate the importance of the residues that interact with the phosphoribosyl group of OMP for transition-state stabilization and, especially, the catalytic tetrad for initiation and decarboxylation. The tetrad consists of two negatively charged aspartates (Asp312 and Asp317) and two positively charged lysines (Lys281 and Lys314). Their structural arrangement results in an alternating-charge configuration that supports a proton transfer within the tetrad. The associated electrostatic and geometric restrain assures proper substrate binding. This configuration is crucial, as it allows Asp312 to initiate catalysis by forming a low-barrier hydrogen bond with the carboxylate group of OMP. In case the charge configuration is modified, by exchanging Asp312 or Asp317 with alanine and/or asparagine, the formation of hydrogen bonds between the positively charged lysines and the carboxylate group of the substrate is facilitated. As a consequence, the reactant state is stabilized and catalysis inhibited. Further mechanistic implications based on the analysis of 23 variants are discussed in this thesis and enabled an assessment of the overall role of these residues on catalysis. As part of the de novo pyrimidine synthesis, OMPDCase is expressed in all domains of life. Thus, the identification of new inhibitors is desired, as they might specifically inhibit OMPDCase from different parasites. Here, in a second set of characterizations, different OMP analogs were designed and tested and their inhibitory potential was analyzed. Overall, the results presented will facilitate the design of new ligands that both allow to understand the binding motif of OMPDCase and enable the development of new inhibitors in the future.
Keywords: Uridine monophosphate synthase; Transition-state stabilization; Orotidine 5’-monophosphate decarboxylase (OMPDCase); enthalpy-entropic compensation; catalysis; nucleotides; X-ray crystallography
