title: Mechanistic insights into Sky1p, a yeast homologue of the mammalian SR protein kinases.
abstract: The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct. Biol. 8, 176-183]. To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques. While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity. The latter is achieved through a combination of local and distal factors in the protein substrate. The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands. Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis. While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments. These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products. These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate.
authors.affiliation: Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA 92093, USA.
authors.forename: Brandon E
authors.initials: BE
authors.lastname: Aubol
authors.forename: Brad
authors.initials: B
authors.lastname: Nolen
authors.forename: Don
authors.initials: D
authors.lastname: Vu
authors.forename: Gourisankar
authors.initials: G
authors.lastname: Ghosh
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Adams
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014961:X-Ray Diffraction
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C490216:SKY1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D008274:Magnesium