Computational Simulation Of The Activation Center Of Human Extracellular Superoxide Dismutase And Experimental Verification

Human superoxide dismutases (hSODs) are a group of metalloenzymes playing an important role in protection against oxygen free radicals in aerobic organisms. There are three kinds of superoxide dismutase: intracellular superoxide dismutase(CuZnSOD), extracellular superoxide dismutase (EC-SOD) and Mn superoxide dismutase(Mn-SOD). EC-SOD is isolated for the first time from mammal by Stefan L. Marklund. It is the main SOD-isoenzyme in extracellular matrix.(ECM) EC-SOD is a tetrameric secretory glycoprotein with high affinity for heparin sulfate proteoglycans. Each subunit has one coper and one zinc atom. The primiry structure of EC-SOD contains three components. The N-terminal region supports the quaternary structure and contains the consensus for N-linked glycosylation at Asn89. The C-terminal region mediates the binding of EC-SOD to components in extracellular matrix. The central region of human EC-SOD is approximately 50% identical to the final two-thirds of Cu/Zn-SOD. And it contains all the ligands essential for the coordination of the active site Cu(II) and Zn(II) ions. The N-terminal region of EC-SOD is important for the formation of tetramers, and the C-terminal region of hEC-SOD mediates ligands binding in ECM including heparin sulfate proteoglycans and type I collagen . The heparin binding region of EC-SOD is removed by an intracellular proteolytic event before secretion. The size of EC-SOD molecue is too big to be determined structurally by NMR. Indeed difficulties in crystallization of EC-SOD due to its glycosylation lead to the failure in determination of EC-SOD three-dimensional structure by X-ray crystallography.In the first section of this thesis, we reported the construction of a chimeric protein, which was designated as hySOD, comprising the N- and C-terminal domains of human extracellular superoxide dismutase (hEC-SOD) and the central domain of intracellular human CuZnSOD (hCuZnSOD). The recombinant protein can be produced in large quantities in Pichia pastoris, and showed a single band at about 34 kDa by SDS?PAGE. The specific activity of hySOD was 630±20 U/mg, which was proved by activity stain. The enzyme is highly resistant to heat and is reportedly able to protect cells from transformed heat shock and H2O2 oxidative stress. These suggested that the catalytic mechanism of CuZnSOD is similar to that of EC-SOD.Elucidation of EC-SOD structure is essential in understanding the catalytic mechanism of EC-SOD. However, despite numerous efforts, the crystal structure of human EC-SOD has not been determined.In the second section of this thesis, we constructed in silico aEC-SOD and iEC-SOD through homology modeling based on the structure of CuZnSOD. Molecular dynamics simulations were then performed to explore the interactions between the O2●- and aEC-SOD. Through comparison between the inteaction of O2●- to aEC-SOD and the inteaction of O2●- to iEC-SOD, we revealed the lost-function determinant of iEC-SOD. In addition, we constructed the activation center domain of aEC-SOD and iEC-SOD to investigate the accuracy of the homology modeling. Our results suggested that the central region of aEC-SOD and CuZnSOD exhibited similar structural characteristics. The Cu was ligated to four nitrogen atoms from different His in a distorted trigonal planar coordination geometry. The distance between Arg and Cu is appropriate to form a steady reaction-complex. While the difference of structure of iECSOD is the distorted trigonal planar coordination geometry formed by His is not steady. And The distance between Arg and Cu is changed resulting in strucurally unstable in the reaction-complex. The results from molecular biology and biochemistry studies also confirmed the the accuracy of the homology modeling.