Abstract
S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His6-S6K1αII(ΔAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (KdATP = 5-6 μM) was followed by low affinity binding of Tide (KdTide = 180 μM), and values of KmATP = 5-6 μM and K mTide = 4-5 μM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.
| Original language | English |
|---|---|
| Pages (from-to) | 11972-11980 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 283 |
| Issue number | 18 |
| DOIs | |
| State | Published - May 2 2008 |
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ASJC Scopus subject areas
- Biochemistry
- Cell Biology
- Molecular Biology
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Kinetic mechanism of fully activated S6K1 protein kinase. / Keshwani, Malik M.; Harris, Thomas K.
In: Journal of Biological Chemistry, Vol. 283, No. 18, 02.05.2008, p. 11972-11980.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Kinetic mechanism of fully activated S6K1 protein kinase
AU - Keshwani, Malik M.
AU - Harris, Thomas K
PY - 2008/5/2
Y1 - 2008/5/2
N2 - S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His6-S6K1αII(ΔAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (KdATP = 5-6 μM) was followed by low affinity binding of Tide (KdTide = 180 μM), and values of KmATP = 5-6 μM and K mTide = 4-5 μM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.
AB - S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His6-S6K1αII(ΔAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (KdATP = 5-6 μM) was followed by low affinity binding of Tide (KdTide = 180 μM), and values of KmATP = 5-6 μM and K mTide = 4-5 μM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.
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U2 - 10.1074/jbc.M800114200
DO - 10.1074/jbc.M800114200
M3 - Article
VL - 283
SP - 11972
EP - 11980
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 18
ER -