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Kinetic analysis of the cyclin-dependent kinase-activating kinase (Cak1p) from budding yeast

Enke, Deborah A. ; Kaldis, Philipp LU orcid and Solomon, Mark J. (2000) In Journal of Biological Chemistry 275(43). p.33267-33271
Abstract

Cak1p, the Cyclin-dependent kinase-activating kinase from budding yeast, is an unusual protein kinase that lacks many of the highly conserved motifs observed among members of the protein kinase superfamily. Cak1p phosphorylates and activates Cdc28p, the major cyclin-dependent kinase (CDK) in yeast, and is thereby required for passage through the yeast cell cycle. In this paper, we explore the kinetics of CDK phosphorylation by Cak1p, and we examine the role of the catalytic step in the reaction mechanism. Cak1p proceeds by a sequential reaction mechanism, binding to both ATP and CDK2 with reasonable affinities, exhibiting K(d) values of 7.2 and 0.6 μM, respectively. Interestingly, these values are approximately the same as the K(M)... (More)

Cak1p, the Cyclin-dependent kinase-activating kinase from budding yeast, is an unusual protein kinase that lacks many of the highly conserved motifs observed among members of the protein kinase superfamily. Cak1p phosphorylates and activates Cdc28p, the major cyclin-dependent kinase (CDK) in yeast, and is thereby required for passage through the yeast cell cycle. In this paper, we explore the kinetics of CDK phosphorylation by Cak1p, and we examine the role of the catalytic step in the reaction mechanism. Cak1p proceeds by a sequential reaction mechanism, binding to both ATP and CDK2 with reasonable affinities, exhibiting K(d) values of 7.2 and 0.6 μM, respectively. Interestingly, these values are approximately the same as the K(M) values, indicating that the binding of substrates is fast with respect to catalysis and that the most likely reaction mechanism is rapid equilibrium random. Cak1p is a slow enzyme, with a catalytic rate of only 4.3 min -1. The absence of a burst phase indicates that product release is not rate-limiting. This result, and a solvent isotope effect, suggests that a catalytic step is rate-limiting.

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author
; and
publishing date
type
Contribution to journal
publication status
published
in
Journal of Biological Chemistry
volume
275
issue
43
pages
5 pages
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • pmid:10934199
  • scopus:0034721935
ISSN
0021-9258
DOI
10.1074/jbc.M004748200
language
English
LU publication?
no
id
d0802ea1-228e-45ef-b162-f155a6740fd1
date added to LUP
2019-09-18 14:31:01
date last changed
2024-03-19 21:35:19
@article{d0802ea1-228e-45ef-b162-f155a6740fd1,
  abstract     = {{<p>Cak1p, the Cyclin-dependent kinase-activating kinase from budding yeast, is an unusual protein kinase that lacks many of the highly conserved motifs observed among members of the protein kinase superfamily. Cak1p phosphorylates and activates Cdc28p, the major cyclin-dependent kinase (CDK) in yeast, and is thereby required for passage through the yeast cell cycle. In this paper, we explore the kinetics of CDK phosphorylation by Cak1p, and we examine the role of the catalytic step in the reaction mechanism. Cak1p proceeds by a sequential reaction mechanism, binding to both ATP and CDK2 with reasonable affinities, exhibiting K(d) values of 7.2 and 0.6 μM, respectively. Interestingly, these values are approximately the same as the K(M) values, indicating that the binding of substrates is fast with respect to catalysis and that the most likely reaction mechanism is rapid equilibrium random. Cak1p is a slow enzyme, with a catalytic rate of only 4.3 min <sup>-1</sup>. The absence of a burst phase indicates that product release is not rate-limiting. This result, and a solvent isotope effect, suggests that a catalytic step is rate-limiting.</p>}},
  author       = {{Enke, Deborah A. and Kaldis, Philipp and Solomon, Mark J.}},
  issn         = {{0021-9258}},
  language     = {{eng}},
  month        = {{10}},
  number       = {{43}},
  pages        = {{33267--33271}},
  publisher    = {{American Society for Biochemistry and Molecular Biology}},
  series       = {{Journal of Biological Chemistry}},
  title        = {{Kinetic analysis of the cyclin-dependent kinase-activating kinase (Cak1p) from budding yeast}},
  url          = {{http://dx.doi.org/10.1074/jbc.M004748200}},
  doi          = {{10.1074/jbc.M004748200}},
  volume       = {{275}},
  year         = {{2000}},
}