title: Isoleucine 69 and valine 325 form a specificity pocket in human muscle creatine kinase.
abstract: Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by ATP. From a structural perspective, the enzyme utilizes two flexible loop regions to sequester and position the substrates for catalysis. There has been debate over the specific roles of the flexible loops in substrate specificity and catalysis in CK and other related phosphagen kinases. In CK, two hydrophobic loop residues, I69 and V325, make contacts with the N-methyl group of creatine. In this study, we report the alteration of the substrate specificity of CK through the mutagenesis of V325. The V325 to glutamate mutation results in a more than 100-fold preference for glycocyamine, while mutation of V325 to alanine results in a slight preference of the enzyme for cyclocreatine (1-carboxymethyl-2-iminoimidazolidine). This study enhances our understanding of how the active sites of phosphagen kinases have evolved to recognize their respective substrates and catalyze their reactions.
authors.affiliation: Department of Biopharmaceutical Sciences, University of California, 600 16 Street, San Francisco, California 94143, USA.
authors.forename: Walter R P
authors.initials: WR
authors.lastname: Novak
authors.forename: Pan-Fen
authors.initials: PF
authors.lastname: Wang
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McLeish
authors.forename: George L
authors.initials: GL
authors.lastname: Kenyon
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Babbitt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry; D002384:Catalysis; D003402:Creatine Kinase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003404:Creatinine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D013045:Species Specificity; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D014633:Valine / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D007532:Isoleucine; C012260:cyclocreatine; D003404:Creatinine; D003402:Creatine Kinase; D001122:Arginine Kinase; C004946:glycocyamine; D014633:Valine; D005998:Glycine