Steady-state kinetic mechanism of PDK1

Xinxin Gao, Thomas K Harris

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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(ΔPH)). To gain further insight to PIF-dependent catalysis, steady-state kinetic and inhibition studies were performed for His6-PDK1(ΔPH)-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 (αKdATP = 40 μM and αKdTide = 80 μM) from individual transitory binary complexes (KdATP = 0.6 μM and Kd Tide = 1 μM). 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, ADPand 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 kcat 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 ≤ η/η0 ≤ 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.

Original languageEnglish
Pages (from-to)21670-21681
Number of pages12
JournalJournal of Biological Chemistry
Volume281
Issue number31
DOIs
StatePublished - Aug 4 2006

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Tides
Phosphorylation
Kinetics
Consensus Sequence
Phosphotransferases
Catalysis
Adenosine Diphosphate
Substrates
Adenosine Triphosphate
Phosphates
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Steady-state kinetic mechanism of PDK1. / Gao, Xinxin; Harris, Thomas K.

In: Journal of Biological Chemistry, Vol. 281, No. 31, 04.08.2006, p. 21670-21681.

Research output: Contribution to journalArticle

Gao, Xinxin ; Harris, Thomas K. / Steady-state kinetic mechanism of PDK1. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 31. pp. 21670-21681.
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abstract = "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(ΔPH)). To gain further insight to PIF-dependent catalysis, steady-state kinetic and inhibition studies were performed for His6-PDK1(ΔPH)-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 (αKdATP = 40 μM and αKdTide = 80 μM) from individual transitory binary complexes (KdATP = 0.6 μM and Kd Tide = 1 μM). 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, ADPand 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 kcat 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 ≤ η/η0 ≤ 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.",
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