Theoretical Studies On The Mechanisms Of Several Important Enzymatic Reactions

Enzymes are biological catalysts. Most enzymes are proteins, although some catalytic RNA molecules have been identified. Due to their different specificity, they could be classified to several categories, including oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases. Enzymes are responsible for the thousands of chemical reactions that sustain life. With the development of science and technology, more and more enzymes are applied in our daily life. Although some information such as the crystal structures, the catalytic activity, activating agent, inhibitors, could be obtained through the experimental methods, the details of their catalytic mechanisms are not sure. Computer simulations are able to give the detailed information of the enzymatic reactions on the atomic level.In this dissertation, we use quantum mechanics (QM), molecular mechanics (MM), and combined quantum mechanics and molecular mechanics (QM/MM) methods to study several important enzymatic reactions.The main contents as follow:(1) The mechanism of isomerase—phenylalanine aminomutase.The Taxus canadensis phenylalanine aminomutase (TcPAM) catalyzes the isomeriazation of (S)-a-phenylalanine to the (R)-β-isomer. The active site of TcPAM contains the signature5-methylene-3,5-dihydroimidazol-4-one (MIO) prosthesis, observed in the ammonia lyase class of enzymes. An enchanting place of PAM is that the product of PAM is also an obligatory biosynthetic precursor of the phenylisoserine side chain of antimitotic pharmaceutical Taxol. Up to now, there are two plausible mechanisms for these MIO-dependent enzymes, i.e., the amino-MIO adduct mechanism and the Friedel-Crafts-type reaction mechanism. In response to this mechanistic uncertainty, the phenylalanine aminomutase mechanism was investigated by using density functional methods. The main results can be summarized as follows: The reaction proceeds through an amino-MIO adduct mechanism, but not a Fricdel-Crafts-type mechanism. In the amino-MIO adduct mechanism, the deprotonation at the (3-position and ammonia elimination occur on the amino-MIO adduct through an ElcB mechanism. The stereochemistry of the TcPAM reaction can be achieved by rotation of the intermediate cinnamate round the Cl-Cα bond prior to rebinding of the amino group at the β-position on the intermediate. This would be the reason that TcPAM catalyzes the opposite stereochemistry production compared with PaPAM and SgTAM. The role of several important active-site residues are illustrated according to our calculations. The mechanism described here for TcPAM is consistent with several experimental results, and provides strong theoretical support for the stereochemistry. This is expected to shed light on the preparation of the chiral building blocks and the biosynthetic engineering toward novel therapeutics.(2) The catalytic mechanisms of hydrolascs—poly(ADP-ribose) glycohydrolase and limonene1,2-epoxide hydrolase.Poly(ADP-ribose) glycohydrolase (PARC) is the only enzyme responsible for the degradation of ADP-ribose polymers. PARC activity is critical for the prevention of poly(ADP-ribose) polymerase (PARP) dependent cell death by regulating the intracellular levels of PAR. Very recently, the first crystal structure of PARG was reported (Dea Slade, et al, Nature477(2011)616), and a possible SN1-type-like mechanism was proposed. Apart from these observations, little is known about the PAR degrading reaction. Our calculations confirm that PARG catalyzes the hydrolysis of PAR via an SN2-like mechanism. By using different active site models, the roles of key residues have been illustrated. Glu115works as a proton donor to the glycosidic oxygen as well as a proton acceptor from the activated water molecule. The other glutamic residue Glu114plays an important role in stabilizing ribose" by forming a hydrogen bond to the C2"-OH of ribose". The negative diphosphate group significantly lowers the reaction barrier by providing a strong hydrogen bond to the water molecule and influencing the sterie orientation of ribose". And Phe227provides the spacial effects to position the terminal ribose". Our mechanism picture described here is expected to present a versatile paradigm of the mechanisms of ribose-ribose O-glycosidic bond hydrolysis.Limonene1,2-epoxide hydrolase (LEH) is completely different from those of classic epoxide hydrolases (EHs) which catalyze the hydrolysis of epoxides to vicinal diols. A novel concerted general acid catalysis step involving the Asp101-Arg99-Asp132triad is proposed to play an important role in the mechanism. The detailed mechanism of epoxide ring-opening catalyzed by limonene1,2-epoxide hydrolase has been studied by combined QM/MM methodologies. The calculations indicate that the LEH-catalyzed hydrolysis proceeds via a novel single-step conceited general acid reaction mechanism. The reaction path is demonstrated to involve simultaneous donation of an Asp101proton to the epoxide oxygen, nucleophilic attack of water at the more substituted oxirane carbon, and abstraction of a proton from water by Asp132. Our QM/MM calculations give an energy barrier of16.9kcal/mol for the formation of limonene-1,2-diol by nucleophilic attack on the more substituted epoxide carbon. According to the QM/MM optimized structures and minimized energy profiles for mutagenesis, the proximal protein environment, especially the positions of Arg99, Tyr53and Asn55, plays an important role in the LEH-mediated catalytic reaction.(3) The catalytic mechanism of oxidoreductase—pyruvate decarboxylase.Pyruvate decarboxylase (PDC) is a typical thiamin diphosphate (ThDP)-dependent enzyme with widespread applications in industry. Though studies regarding the reaction mechanism of PDC have been reported, they are mainly focused on the formation of ThDP ylide and some elementary steps in the catalytic cycle, studies about the whole catalytic cycle of PDC are still not completed. In these previous studies, a major controversy is whether the key active residues (Glu473, Glu50’, Asp27’, His113’, His114’) are protonated or ionized during the reaction. To explore the catalytic mechanism and the role of key residues in the active site, three whole-enzyme models were considered and the combined QM/MM calculations on the nonoxidative decarboxylation of pyruvate to acetaldehyde catalyzed by PDC were performed. According to our computational results, the fundamental reaction pathways, the complete energy profiles of the whole catalytic cycle, and the specific role of key residues in the common steps were obtained. It is also found that the same residue with different protonation states will lead to different reaction pathways and energy profiles. The mechanism derived from the model in which the residues (Glu473, Glu50’, Asp27’, His113’, Hisl14’) are in their protonated states is most consistent with experimental observations. Therefore, extreme care must be taken when assigning the protonation states in the mechanism study.(4) The catalytic mechanisms of transferases—O6-alkylguanine-DNA alkyltransferase and phenylethanolamine N-methyltransferase.DNA alkylation can be caused from both endogenous and exogenous DNA-damaging agents, such as S-adenosylmethionine in the cell, methylmethane sulfonate (MMS) or N-methyl-N’-nitro-N-nitroso-guanidine (MNNG) in the environment. Alkylation adducts frequently occur at O6-position of guanine, resulting to O6-methylguanine (O6-mG), which if not repaired leads to GC to AT transition mutations and cancer. The combined quantum-mechanical/molecular-mechanical (QM/MM) approaches have been applied to investigate the detailed reaction mechanism of human06-alkylguanine-DNA alkyltransferase (AGT). AGT is a direct DNA repair protein, which is capable of repairing the alkylated DNA by transferring the methyl group to the thiol group of cysteine residue (Cys145) in the active site in an irreversible and stoichiometric reaction. Our QM/MM calculations reveal that the methyl group transferring step is expected to occur through two steps, in which the methyl carbocation generating step is the rate-determining step with an energy barrier of14.4kcal/mol at QM/MM B3LYP/6-31G(d, p)//CHARMM22level of theory. It is different from the previous theoretical studies based on QM calculations by using cluster model that the methyl group transferring step is a one-step process with higher energy barrier.Epinephrine is a naturally occurring adrenomedullary hormone that transduces environmental stressors into cardiovascular actions. As the only route in the catecholamine biosynthetie pathway, Phenylethanolamine N-methyltransferase (PNMT) catalyzes the synthesis of epinephrine. To elucidate the detailed mechanism of enzymatic catalysis of PNMT, combined quantum-mechanical/molecular-mechanical (QM/MM) calculations were performed. The calculation results reveal that this catalysis contains three elementary steps:the deprotonation of protonated norepinphrine, the methyl transferring step and deprotonation of the methylated norepinphrine. The methyl transferring step was proved to be the rate-determining step undergoing a SN2mechanism with an energy barrier of16.4kcal/mol. During the whole catalysis, two glutamic acids Glu185and Glu219were proved to be loaded with different effects according to the calculations results of the mutants. These calculation results can be used to explain the experimental observations and make a good complementarity for the previous QM study.(5) The mechanism of biofunctional enzyme—fructose-1,6-bisphosphate aldolase/phosphatase (FBPA/P).Arc/weal fructose-1,6-bisphosphale (FBP) aldolase/phosphalase (FBPA/P) is a bifunctional enzyme that catalyses two chemically distinct reactions of gluconeogenesis:(a) the reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) to FBP;(b) the dephosphorylation of FBP to fructose-6-phosphate (F6P). Thus, FBPA/P is fundamentally different from ordinary enzymes whose active sites are responsible for a specific reaction. There are very less studies focus on the catalytic mechanism of FBPA/P. We have investigated the mechanism of FBPA/P using QM/MM methods. Our results agree well with the experiments that the Schiff base was obtained as one intermediate during the reaction process. Formation of C3-C4bond of FBP is the rate-limiting step and undergoes a free energy barrier of24.7kcal/mol. Lys232formed a Schiff base intermediate with the substrate of DHAP. Try229could sever as the catalytic acid/base residue for all the steps. Study on the simulation of the active-site remodeling in FBPA/P is ongoing.