HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 31, 21670-21681, August 4, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/31/21670    most recent M602448200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gao, X. Articles by Harris, T. K. Search for Related Content PubMed PubMed Citation Articles by Gao, X. Articles by Harris, T. K. Social Bookmarking                      What's this?

Steady-state Kinetic Mechanism of PDK1^*

Xinxin Gao and Thomas K. Harris¹

From the Department of Chemistry, University of Miami, Coral Gables, Florida 33124 and the Department of Biochemistry and Molecular Biology, University of Miami, Miller School of Medicine, Miami, Florida 33136

PDK1 catalyzes phosphorylation of Thr in the conserved activation loop region of a number of its downstream AGC kinase family members. In addition to the consensus sequence at the site of phosphorylation, a number of PDK1 substrates contain a PIF sequence (PDK1-interacting fragment), which binds and activates the kinase domain of PDK1 (PDK1(PH)). To gain further insight to PIF-dependent catalysis, steady-state kinetic and inhibition studies were performed for His₆-PDK1(PH)-catalyzed phosphorylation of PDK1-Tide (Tide), which contains an extended "PIF" sequence C-terminal to the consensus sequence for PDK1 phosphorylation. In two-substrate kinetics, a large degree of negative binding synergism was observed to occur on formation of the active ternary complex ( and ) from individual transitory binary complexes ( and ). On varying ATP concentrations, the ADP product and the (T/E)-PDK1-Tide product analog (p'Tide) behaved as competitive and noncompetitive inhibitors, respectively; on varying Tide concentrations, ADP and p'Tide behaved as noncompetitive and competitive inhibitors, respectively. Also, negative binding synergism was associated with formation of dead-end inhibited ternary complexes. Time progress curves in pre-steady-state studies under "saturating" or k_cat conditions showed (i) no burst or lag phenomena, (ii) no change in reaction velocity when adenosine 5'-O-(thiotriphosphate) was used as a phosphate donor, and (iii) no change in reaction velocity on increasing relative microviscosity (0 /₀ 3). Taken together, PDK1-catalyzed trans-phosphorylation of PDK1-Tide approximates a Rapid Equilibrium Random Bi Bi system, where motions in the central ternary complex are largely rate-determining.

Received for publication, March 15, 2006 , and in revised form, May 4, 2006.

^* This work was supported by the James and Esther King Biomedical Research Program (BM026) and National Institutes of Health NIGMS Grant GM69868 (to T. K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Miami, Miller School of Medicine, P. O. Box 016129, Miami, FL 33101-6129. Tel.: 305-243-3358; Fax: 305-243-3955; E-mail: tkharris{at}miami.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?