Dual-specific Cdc25B phosphatase: in search of the catalytic acid.

Published

Journal Article

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly structurally homologous to other well-characterized members, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. However, the catalytic acid needed for protonation of the leaving group has yet to be identified. To elucidate the role and identity of this key catalytic residue, we have performed a detailed pH-dependent kinetic analysis of Cdc25B. The pK(a) of the catalytic cysteine was found to be 5.6-6.3 in steady state and one-turnover burst experiments using the small molecule substrates p-nitrophenyl phosphate and 3-O-methylfluorescein phosphate. Interestingly, Cdc25B does not exhibit the typical bell-shaped pH-rate profile with small molecule substrates seen in other cysteine phosphatases and indicative of the catalytic acid because it lacks pH dependence between 6.5 and 9. Reactions of Cdc25B with the natural substrate Cdk2-pTpY/CycA, however, did yield a bell-shaped pH-rate profile with a pK(a) of 6.1 for the catalytic acid residue. Recent structural studies of Cdc25 have suggested that Glu474 [Fauman, E. B., et al. (1998) Cell 93, 617-625] or Glu478 [Reynolds, R. A., et al. (1999) J. Mol. Biol. 293, 559-568] could function as the catalytic acid in Cdc25B. Using site-directed mutagenesis and truncation experiments, however, we found that neither of these residues, nor the unstructured C-terminus, is responsible for the observed pH dependence. These results indicate that the catalytic acid does not appear to lie within the known structure of Cdc25B and may lie on its protein substrate.

Full Text

Duke Authors

Cited Authors

  • Chen, W; Wilborn, M; Rudolph, J

Published Date

  • September 5, 2000

Published In

Volume / Issue

  • 39 / 35

Start / End Page

  • 10781 - 10789

PubMed ID

  • 10978163

Pubmed Central ID

  • 10978163

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi000909u

Language

  • eng

Conference Location

  • United States