title: Changing the substrate specificity of creatine kinase from creatine to glycocyamine: evidence for a highly evolved active site.
abstract: Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.
authors.affiliation: Department of Chemistry, College of Wooster, 943 College Mall, Wooster, OH 44691, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Jourden
authors.forename: Callisia N
authors.initials: CN
authors.lastname: Clarke
authors.forename: Allyson K
authors.initials: AK
authors.lastname: Palmer
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Barth
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Prada
authors.forename: Robyn N
authors.initials: RN
authors.lastname: Hale
authors.forename: Dean
authors.initials: D
authors.lastname: Fraga
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Snider
authors.forename: Paul L
authors.initials: PL
authors.lastname: Edmiston
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003401:Creatine / Q000378:metabolism*; D052283:Creatine Kinase, MM Form / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019143:Evolution, Molecular; D015870:Gene Expression; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D052283:Creatine Kinase, MM Form; C004946:glycocyamine; D003401:Creatine; D005998:Glycine