Mechanistic insights into Sky1p, a yeast homologue of the mammalian SR protein kinases.
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.
Aubol
Brandon E
BE
Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA 92093, USA.
Nolen
Brad
B
Vu
Don
D
Ghosh
Gourisankar
G
Adams
Joseph A
JA
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
D029701:Saccharomyces cerevisiae Proteins; C490216:SKY1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D008274:Magnesium