Web Release Date: July 9,
Mechanistic Insights into Sky1p, a Yeast Homologue of the Mammalian SR Protein
Kinases
and
Department of Chemistry and Biochemistry and Department of Pharmacology, University of California, San Diego, La Jolla, California, 92093
Received March 26, 2002
Revised Manuscript Received June 6, 2002
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 Mg2+-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.
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