Hydrolysis and Cyanolysis of DTNB-Modified Creatine Kinase

The cyanolysis of DTNB-modified creatine kinase(S,S’-di-TNB-CK)has been studied.Itwas found that there exist both cyanolysis and hydrolysis at the same time under the cyanolysis condition de-scribed previously by Degani.DTNB-modified creatine kinase was rapidly hydrolyzed at pH 9.5 in the ab-sence of KCN.The hydrolysis shows biphasic kinetics as seen in the semilogarithmic pseudo-first-order rateplot.The analysis shows that about one S-TNB group/mol of DTNB-modified creatine kinase was rapidly re-leased in the fast phase of the hydrolysis reaction.The further cyanolysis of hydrolytic products showsmonophasic kinetics,and about one S-TNB group/mol of hydrolytic product was also rapidly released.Theabove results show that one of the two TNB-labeled thiol groups situated respectively at two active sites ofthe creatine kinase molecule was rapidly hydrolyzed,and the other was hydrolyzed at a very slow rate.Whenthe hydrolyzed products were cyanolyzed,the other residual TNB group was also released.These resultssuggest that the subunits of creatine kinase are asymmetrically associated.This leads to the differential envi-ronments of the two thiol groups at the active sites of two subunits.The above results also show that withthe TNB release during hydrolysis or cyanolysis,the enzymic activity was also partially recovered at the sametime.The recovery in activity is linearly related to the extent of the regeneration of reactive thiol groups.Therefore,it is suggested that the reactive thiol groups of enzyme are essential for its activity,and they arelocated in the active sites of dimeric enzyme.