The Study Of The Polar Microenvironment Arround The Reactive Cys283 In CK, As Well As The Inactivation And The Structural Changes

Found in all vertebrates, creatine kinase (CK;ATP: creatine N-phosphotransferase;EC 2.7.3.2) catalyzes the reversible transfer of a phosphoryl group from MgATP to creatine, producing phosphocreatine and MgADP. CK plays an important role in the energy homeostasis of cells with intermittently high energy requirements. The enzyme is of clinical importance and its levels are routinely used as an indicator of myocardial and skeletal muscle disorders and for the diagnosis of acute myocardial infarction.Recently, studies have shown that inactivation occurred before noticeable conformational change during CK denaturation. More results proved that the active sites of the enzyme displayed more conformational flexibility. CK have five essential amino acid residues, Histidine, Lysine, Arginine, Tryptophan and Cysteine. One highly reactive cysteine, identified as Cys283 in rabbit muscle CK is fully conserved in the CKs. The X-ray structure of Mi_b-CK showed that Cys283 was indeed located within the active site and was positioned near the γ-phosphate of ATP and near the acidic residues thought to be involved in creatine binding.In this study, we prepared the rabbit muscle CK as described by Yao, firstly. Then, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 6,6'-dithiodinicotinic acid (DTNA) and 2,2'-dithiodipyridine (DSDP) were used as inhibitors for the exploration of the polar microenvironment around the Cys283 in CK. Complete kinetics of inactivation was monitored, and the relevant microscopic rate constants of the reagents with the various enzyme-substrate complexes were also determined. The result show that the inactivation of CK by 5,5'-dithiobis(2-nitrobenzoic acid) was fastest, followed by 6,6'-dithiodinicotinic acid and then 2,2'-dithiodipyridine, which appears that the electrophilic group is predominant around the Cys283. Otherwise, oxidized-DTT was also adopted as a modifier in CK oxidization, in order to study the effect of oxidiation on the catalysis and conformational change of creatine kinase from rabbit muscle.