RABBIT MUSCLE CREATINE-KINASE - CONSEQUENCES OF THE MUTAGENESIS OF CONSERVED HISTIDINE-RESIDUES

Creatine kinase (CK; EC 2.7.3.2) catalyzes the reversible conversion o f creatine and MgATP to phosphocreatine and MgADP. In the absence of a n X-ray crystal structure, we have used the sequence homology of creat ine kinases and other guanidino kinases from a variety of sources to i dentify the conserved histidine residues in rabbit muscle CK, as well as to try to pinpoint a reactive histidine that has been implicated in the active site. This residue has been proposed to act as a general a cid/base catalyst assisting in the phosphoryl transfer mechanism [Cook et al. (1981) Biochemistry 20, 1204-1210]. There are 17 histidine res idues in rabbit muscle CK, and of these, only five have been conserved in all guanidino kinase sequences published to date [Muhlebach et al. (1994) Mel. Cell. Biochem. 133, 245-62]. In rabbit muscle CK, these r esidues are H96, H105, H190, H233, and H295. We have carried out site- specific mutagenesis of these five histidine residues, replacing each with an asparagine. Each of these mutants exhibited enzymatic activity but to varying degrees. The H105N, H190N, and H233N mutants displayed specific activities similar to that of the wild-type enzyme. H96N has high activity, but appears to be quite unstable, losing catalytic act ivity upon cell lysis by sonication and/or chromatographic steps invol ved in purification. H295N shows a significantly reduced catalytic act ivity relative to the native enzyme, due to marked decreases in k(cat) and the affinities for both substrates. Each of the five mutants is i nactivated by diethyl pyrocarbonate (DEP), and inactivation is reversi ble upon incubation with hydroxylamine. However, only H295N shows a dr amatically reduced rate of inactivation relative to native CK, consist ent with H295 being the residue modified by DEP in the native enzyme. These intriguing results indicate that four of the conserved histidine s (H96, H105, H295, and H233) are not essential for activity, and whil e H295 may be at the active site of CK, it is unlikely to play the rol e of a general acid/base catalyst.