AbstractsChemistry

Trans-sialidase is a vital enzyme for the lifecycle of Trypanosoma cruzi, the protozoa responsible for Chagas' disease, which is lethal and drastically affects large human populations in Central and South America, widening its epidemic area to North America in recent years. T. cruzi trans-sialidase (TcTS) catalyzes transfer of sialic acids from host glycoconjugates to the parasite's glycoconjugates, which facilitates the parasite the means to escape from the host immune system and to invade the host cells. Thus, TcTS stands as a potential and appealing therapeutic target for Chagas' disease. Experimental evidence suggests that a relatively long-lived covalent intermediate forms in the mechanism of TcTS. If this scenario is correct, sialic acid is scavenged from the host's glycoconjugates and stays bound to the enzyme until the parasite's glycoconjugate enters the active site. However, it is unclear whether the covalent intermediate formation occurs through an SN1 or SN2 mechanism. It is crucial to elucidate the mechanism and the transition structure for future inhibitor design studies of TcTS. Additionally, the common inhibitors for sialidases, which catalyze hydrolysis of sialic acids, do not work for TcTS. The reason for this is unclear since both enzyme families share the first step of the mechanism. Trypanosoma rangeli sialidase (TrSA) stands out among sialidases to perform a comparative study with TcTS due to their distinct structural similarity (%70 sequence identity and C-alpha RMSD of 0.59 ?) and yet, different catalytic function. There is experimental evidence about formation of a covalent intermediate in TrSA as well, but only for an activated ligand. Thus, there is a possibility that the mechanism of TrSA is artificially biased towards covalent intermediate formation due to the effect of substituents on the natural ligand. Elucidating the difference in mechanisms of TcTS and TrSA could also pave the way to tailor sialidases into trans-sialidases (and glycosidases into trans-glycosidases) to use for efficient synthesis of molecules that currently require long and low-yield chemical processes. In this study, the mechanisms of both enzymes are investigated using two different QM/MM methods in Chapters 3 and 4. Potential energy surfaces are constructed for each enzyme by performing constrained minimizations. Based on the potential energy surfaces, the difference in the mechanisms of the two enzymes is discussed. Furthermore, 50-ns long molecular dynamics simulations are performed for the two enzymes in free, ligand-bound and inhibitor-bound forms and these simulations are analyzed thoroughly in Chapter 5 to distinguish any structural or dynamical differences between the two enzymes and to shed light on the reason of difference in their inhibitor binding ability.