Studies On Inhibitions Of Arginine Kinase And Creatine Kinase In Phosphagen Kinase Family

Phosphagen kinases are not only in animals generally,but contribute to metabolism,storage and utilization of the bioenergy significantly.In recent years, studies on inhibition of phosphagen kinases have been paid close attention to more and more.The members of phosphagen kinase family coming from different species have become the targets for research of enzyme inhibition.Therefore,studies on the structure and function,and the mechanism of inhibition with phophagen kinases, especially arginine kinase(AK) and creatine kinase(CK),can make people(1) understand the metabolic process of energy in organisms more deeply;(2) screen effective inhibitorsto aim directly at AK in harmful insects,and offer a new pathway; (3)study the mechanism of inhibition of CK as a phosphagen kinase during the developing process of some diseases,such as ischemia,neurodegenerative disease, male infertility,muscle functional impairment,etc.Phosphagen(guanidino) kinases are a family of highly conserved enzymes catalyzing the reversible transfer of phosphate from phosphagens such as creatine phosphate,which is catalyzed by creatine kinase(ATP:creatine phosphotransferase, EC 2.7.3.2),and phosphoarginine,which is catalyzed by arginine kinase(ATP: arginine phosphotransferase,EC 2.7.3.3).In the present investigation,the inhibition kinetics of arginine kinase(AK) by Ag⁺,which is one of important heavy metal ions in nature,and the effects of acrylamide on creatine kinase(CK) were studied. Meanwhile,the effect of zinc ions on the activity of HBCK was still studied.And the effects of osmolytes(glycine and proline) on the aggregation of HBCK induced by the zinc ions have been examined.Arginine kinase was isolated and purified from Chinese Shrimp (Fenneropenaeus chinensis) according to a previous report,with some modifications. The sample was purified by Sephacryl S-300 chromatography and DEAE-Sepharose Fast Flow chromatography.The purified enzyme was found to be homogenous by SDS-gel electrophoresis.This enzyme is monomer with a molecular weight of 43,000 Da as determined by SDS-PAGE and Sephadex G-150 chromatography.The K_m(ATP) and K_m(Arg) were 2.38 mM and 2.62 mM respectively.The enzyme exhibited optimum activity at pH 8.2.The effects of Ag⁺ on arginine kinase(AK) from Fenneropenaeus chinensis were studied.Ag⁺ inactivated the activity of AK in a dose dependent manner(IC₅₀= 15μM).Kinetic studies showed that the inactivation of AK by Ag⁺ was reversible and occurred in a noncompetitive inhibition manner(K_i=2.8μM). Spectrofluorimetry results showed that Ag⁺ did not induce conspicuous tertiary structural changes in AK at the corresponding concentration ranges of inactivation studies.However,the secondary structure measured by circular dichroism was slightly changed by Ag⁺.Taken together,these data suggest that the active site of AK is flexible,with the complete loss of activity occurring prior to significant changes in overall structures.Our study provides important insight into the inhibitory mechanism of Ag⁺ on AK and increases our understanding of the influence of Ag⁺ on the mechanism of this metabolic enzyme.Acrylamide is widely used in industry and can be produced by the cooking and processing of foods.It has been found to be harmful to human beings,and human brain CK(HBCK) has been proposed to be one of the important targets of acrylamide.In this thesis,we studied the effects of acrylamide on HBCK activity, structure and the potential binding sites,with such methods as determination of enzyme activity,spectrofluorimetry and molecular simulation.Compared to CKs from rabbit(RBCK),HBCK was fully inactivated at a several-fold lower concentration of acrylamide,and behaved distinct properties upon acrylamide-induced inactivation and structural changes.The binding sites of acrylamide were located at the cleft between the N-and C-terminal domains of CK, and Glu232 was one of the key binding residues.The effects of acrylamide on CK were proposed to be isoenzyme-specific and species-specific,and the underlying molecular mechanisms were discussed.The effect of zinc ions on the activity of HBCK was studied.The results showed that the low concentration of zinc ions will inhibit the activity of the enzyme. The high concentration of zinc ions will induce the enzyme to aggregate.The aggregation of the enzyme depends on the temperature,the enzyme concentration and the zinc ion concentration.The effects of osmolytes(glycine and proline) on the aggregation of HBCK induced by the zinc ions have been examined.The results showed that glycine and proline can effectively prevent aggregation of the enzyme, and partially assist to recover the conformation and activity of the enzyme.Supra citato,studies have been undretaken on inhibitions of arginine kinase and creatine kinase as two important members in phosphagen kinase family in this thesis.And particular research was taken on changes of the structure and catalytic activity of these two important enzymes in energy metabolism.This work has established a foundation for further study on inhibition mechanism of phosphagen kinases and the future application.