The Impact Of The Water Molecules Of The Nucleoside Monophosphate Catalytic Decarboxylation Of Orotic Acid

Orotidine 5`-phosphate decarboxylase (ODCase,) catalyzes the decarboxylation of orotidine 5`-phosphate (OMP) to form uridine 5`-phosphate in the crucial step of nucleic acid biosynthesis. ODCase is one of the most efficient enzymes. It can increase the rate of decarboxylation by 17 orders of magnitude. ODCase distinguishes itself from other enzymes by the fact that there is no direct participation of cofactors or metal ions, nor formation of a covalent intermediate with a group on the protein. Analyzing the mechanism of ODCase can therefore be an important step in understanding enzyme catalysis on a more general level.In 1995, Wolfenden and Radzicka showed that orotidine 5'-monophosphate decarboxylase(ODCase) was the most proficient enzyme. Since then, the mechanism of catalysis has been widely debated although the study about it has been carrying out for decades.During the past decades, some mechanistic proposals with novel features have been forwarded, such as: concerted O-2 protonation mechanism, C-5 protonation mechanism, concerted C-6 protonation/decarboxylation mechanism, electrostatic stabilization of transition state mechanism, electrostatic stress of the ground state mechanism, O-4 protonation mechanism. Most of these mechanisms are only about the partial functional factors such as O-2,O-4 and C-5 . In addition, the influence of water molecules were considered on the superficial level in the past study.Based on the previous study, several models were designed. The effect of water molecules on the enzyme-catalyzed decarboxylation of Orotidine 5'–Monophosphate was investigated using density functional theory with the B3LYP functional. Barriers and ¹⁵N kinetic isotope effect,¹³C kinetic isotope effect of different models were calculated. Our results indicates that water molecules play an important role in TS stabilization.The main results of this thesis are as follows:Firstly, the model in which O-2 of OMP bond to a protonated water molecule were studied used hybrid Density Functional Theory (B3LYP functional) . The barrier of this model is 24.9kcal/mol. Compared with the barrier 39.6kcal/mol of the model just removing the protonated water molecular. The results showed that the indirect interactions between O-2 of the pyrimidine ring and the positive charged residues through chains of water molecules can lower the barrier height for decarboxylation significantly.Secondly , the effect of water molecules bonding to the O-2 and O-4 of OMP in the same time on the enzyme-catalyzed decarboxylation of Orotidine 5'–Monophosphate were investigated using density functional theory in the same level. Barriers of different models have been calculated and results were compared with the case of water molecules bonding to O-2/O-4 respectively. Our computation indicates that water molecules play an important role in TS stabilization. Our computation also indicates that it is the collective actions of various catalytic factors that make ODCase such a proficient enzyme.Thirdly, several models of the decarboxylation of OMP have been designed and optimized. ¹⁵N kinetic isotope effect of N-1 and ¹³C kinetic isotope effect of C-6 were calculated. The outcome of ¹³C kinetic isotope effect and ¹⁵N kinetic isotope effect consisting with the experimental result show that water molecules playing an important role in the decarboxylation of OMP.