TY - JOUR
T1 - Steady-state kinetic mechanism of PDK1
AU - Gao, Xinxin
AU - Harris, Thomas K.
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2006/8/4
Y1 - 2006/8/4
N2 - 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.
AB - 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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U2 - 10.1074/jbc.M602448200
DO - 10.1074/jbc.M602448200
M3 - Article
C2 - 16737971
AN - SCOPUS:33746822258
VL - 281
SP - 21670
EP - 21681
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 31
ER -