Study On The Structure And Activity Of Alkaline Phosphatase And Modle The DNA Enzyme And MoFeco

All of the material and energy change of the life were carried out under the action of the enzyme,and therefore the research of enzymology is undoubtedly important for understanding the nature of the life.This thesis investigates the progress of the enzyme,the enzyme's catalytic mechanism,research methods and molecular enzymology,functions and application of enzymology.An overview of the structure and function of alkaline phosphatase,restriction endonuclease and nitrogenase.Cobalt is one of essential microelements in many biological processes affecting the activity of many enzymes.Our investigations reveal that Zn²⁺ and Mg²⁺,which are in the active site of native calf intestine alkaline phosphatase(CIP),can be replaced by Co²⁺ in solution directly.The effect of Co²⁺ concentration on the substitute reaction was examined over the ratio of[Co²⁺]/[CIP]from 0:1 to 8:1.The quantity of Zn²⁺ in CIP was decreased during the whole course,but the Mg²⁺ was changed inregularly.A series of active site models of CIP which showing the reaction mechanism were proposed.Comparative study of the pH effection on the reaction showed that the low pH was favorable for Co²⁺ replacing Mg²⁺.To understand how the reaction affect the enzyme,we also solved the secondary structure of CIP for the first time after reacted with Co²⁺ in different condition by the CD spectra.CIP was extensively denatured in 3M guanidine hydrochloride(GdnHCl) solution for 2 h.This research investigated the courses of both the refolding and the reactivation in the absence and presence of La³⁺ ions by the fluorescence,UV,and CD spectra.Under suitable renaturation conditions,about 60-70%of the activity was recovered in the absence and presence of different La³⁺ concentrations.There refolding and the reactivation processes were monophasic after dilution in proper solutions with or without La³⁺.The La³⁺ affected both the reactivation and the refolding courses of unfolded CIP.A comparison of rate constants for the refolding of unfolded CIP with those for recovery of enzyme activity at different La³⁺ concentrations showed that they were synchronized.Both of the two processes speeded up due to increasing the concentrations of La³⁺ ions.This result is very important for the study of the relationship between the structure and function of protein.According to the methord of self-assembled,three new kinds of complexes [Mn(N-methyl-imidazole)₂(μ1,3-N₃)₂],[Cu(DMAP)₂(SCN)₂(DMF)₂]and [Cu(DMAP)2(SCN)₂]were synthesized and characterized by the X-Ray diffraction analysis.Investigated their DNA-binding activity by UV,CD,fluorescence spectra, viscometric titration and voltametric studies.The results show that the Mn-complex bond to CT DNA by a groove mode and the complex of copper bond the CT DNA with a mode of intercalation.The ability to cleavage DNA of these complexes were researched by agarose gel electrophoresis method,and the result show that both of the complexes can cleavage DNA.The complex of copper is better than Mn-complex. This method of DNA cleavage which reaction condition was tender and similar to physiological condition is beneficial to further develop the new technique of DNA cleavage.A high-resolution(1.16(?)) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N,O,or C atom(denoted X) inside the central iron prismane([6Fe]) of the[MoFe₇S₉:homocitrate]FeMo-cofactor (FeMo-co).According to this result,a series of Fe-μ-N-(CO)x,Fe-μ-N-(CO)x-S clusters with the center atom of N and Cp- Mo-(CO)x,Cp- Mo-(CO)x-S were synthesized which prepared for the further research of FeMo-co synthesis.