Changing the substrate specificity of creatine kinase from creatine to glycocyamine: evidence for a highly evolved active site. Biochim Biophys Acta 2007 Dec;1774(12):1519-27
Date
11/03/2007Pubmed ID
17976392DOI
10.1016/j.bbapap.2007.10.001Scopus ID
2-s2.0-36849031248 (requires institutional sign-in at Scopus site) 12 CitationsAbstract
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.
Author List
Jourden MJ, Clarke CN, Palmer AK, Barth EJ, Prada RC, Hale RN, Fraga D, Snider MJ, Edmiston PLAuthor
Callisia N. Clarke MD Chief, Associate Professor in the Surgery department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAnimals
Binding Sites
Creatine
Creatine Kinase, MM Form
Evolution, Molecular
Gene Expression
Glycine
Humans
Models, Biological
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutant Proteins
Phylogeny
Rabbits
Sequence Homology, Amino Acid
Substrate Specificity
