title: Rabbit muscle creatine kinase: consequences of the mutagenesis of conserved histidine residues.
abstract: Creatine kinase (CK; EC 2.7.3.2) catalyzes the reversible conversion of creatine and MgATP to phosphocreatine and MgADP. In the absence of an X-ray crystal structure, we have used the sequence homology of creatine kinases and other guanidino kinases from a variety of sources to identify 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 acid/base catalyst assisting in the phosphoryl transfer mechanism [Cook et al. (1981) Biochemistry 20, 1204-1210]. There are 17 histidine residues in rabbit muscle CK, and of these, only five have been conserved in all guanidino kinase sequences published to date [Mühlebach et al. (1994) Mol. Cell. Biochem. 133, 245-62]. In rabbit muscle CK, these residues 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 activity upon cell lysis by sonication and/or chromatographic steps involved in purification. H295N shows a significantly reduced catalytic activity relative to the native enzyme, due to marked decreases in kcat and the affinities for both substrates. Each of the five mutants is inactivated by diethyl pyrocarbonate (DEP), and inactivation is reversible upon incubation with hydroxylamine. However, only H295N shows a dramatically reduced rate of inactivation relative to native CK, consistent with H295 being the residue modified by DEP in the native enzyme. These intriguing results indicate that four of the conserved histidines (H96, H105, H295, and H233) are not essential for activity, and while H295 may be at the active site of CK, it is unlikely to play the role of a general acid/base catalyst.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
authors.forename: L H
authors.initials: LH
authors.lastname: Chen
authors.forename: C L
authors.initials: CL
authors.lastname: Borders
authors.forename: J R
authors.initials: JR
authors.lastname: Vásquez
authors.forename: G L
authors.initials: GL
authors.lastname: Kenyon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003402:Creatine Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli; D006639:Histidine; D006358:Hot Temperature; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
chemical_list: D006898:Hydroxylamines; D011994:Recombinant Proteins; D019811:Hydroxylamine; D006639:Histidine; D004228:Dithionitrobenzoic Acid; D003402:Creatine Kinase; D004047:Diethyl Pyrocarbonate