Mechanistic insights into Sky1p, a yeast homologue of the mammalian SR protein kinases

被引:18
|
作者
Aubol, BE
Nolen, B
Vu, D
Ghosh, G
Adams, JA [1 ]
机构
[1] Univ Calif San Diego, Dept Pharmacol, La Jolla, CA 92093 USA
[2] Univ Calif San Diego, Dept Chem & Biochem, La Jolla, CA 92093 USA
关键词
D O I
10.1021/bi020233y
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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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收藏
页码:10002 / 10009
页数:8
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