Theoretical Studies Of Metabolic Characteristics And Catalytic Mechanisms Of Catechol-O-methyltransferase And Long Chain L-α-hydroxy Acid Oxidase

In this thesis, we studied the metabolism characteristics and catalytic mechanisms of two types of enzymes, catechol-O-methyltransferase (COMT) and long chain L-a-hydroxy acid oxidase (LCHAO), using theoretical and computational methods.1Catechol-O-methyltransferaseCatechol-O-methyltransferase (COMT, EC2.1.1.6) plays a central role in the inactivation of neurotransmitters sharing a catecholic motif by transferring a methyl group from AdoMet. Methylation of the meta-hydroxyl is much more common than that of the para-hydroxyl in many COMT’s substrates, such as dopamine and norepinephrine. Owing to the catholic parts in quercetin and luteolin, the two important flavonoids are also substrates of COMT. Experimental data showed that quercetin preferred meta-methylation but luteolin favored a para-methylation. To elucidate the mechanism for different preferences of methylations of quercetin and luteolin, we performed theoretical investigation on the different regioseletivities of COMT-catalyzed methylations for quercetin and luteolin by a combined approaches of MD simulations, and QM/MM computations. The binding free energy computation results on the basis of the MD trajectory indicated that quercetin has more stable binding mode for meta-O-methylation than para-O-methylation but luteolin has more stable binding mode for para-O-methylation than meta-O-methylation. On the other hand, our QM/MM computations showed that these two flavonoids have lower reaction energetic barriers for COMT-catalyzed meta-O-methylation than para-O-methylation. We gave a comprehensive explanation considering both thermodynamics and reaction kinetics aspects and discussed the protein-flavonoid interactions as well as the O-methylation processes in our present work. 2Long chain L-α-hydroxy acid oxidaseLong chain L-α-hydroxy acid oxidase (LCHAO) is an FMN-dependent L-α-hydroxy acid oxidase that dehydrogenates L-α-hydroxy acids to corresponding keto-acids. There were two different mechanisms, named as hydride transfer mechanism and carbanion mechanism, respectively, proposed about the catalytic process for FMN-dependent L-α-hydroxy acid oxidase on the basis of biochemical data. However, crystallographic and kinetic studies could not provide enough evidences to prove one of the mechanisms or eliminate the alternative. In the present studies, theoretical computations were carried out to study the molecular mechanism for LCHAO-catalyzed dehydrogenation of L-lactate. Our MD simulations indicated that the binding mode of L-lactate in a crystal structure of LCHAO would be stable for LCHAO-catalyzed oxidation of L-lactate in a hydride transfer mechanism but not a carbanion mechanism. QM/MM calculations were further carried out to obtain the optimized structures of reactants, transition states, and products at the level of ONIOM-EE (B3LYP/6-311++G(d,p)//B3LYP/6-31G(d,p):AMBER). Quantum chemical studies indicated that LCHAO-catalyzed dehydrogenation of L-lactate would be a stepwise catalytic reaction in a hydride transfer mechanism rather than a carbanion mechanism. It was further confirmed that the key residue Tyr129in the active site of LCHAO would not affect L-lactate’s binding to LCHAO but play an important role on the catalytic reaction process through an H-bond interaction by MD simulations, binding free energy calculations, and QM/MM computations on L-lactate binding to the wild-type and Y129F mutant LCHAO, respectively.