Probing the Catalytic Mechanism of the Insulin Receptor Kinase with a Tetrafluorotyrosine-containing Peptide Substrate

Ablooglu, Ararat J.; Till, Jeffrey H.; Kim, Kyonghee; Parang, Keykavous; Cole, Philip A.; Hubbard, Stevan R.; Kohanski, Ronald A.

doi:10.1074/jbc.m003524200

Cited by 32 publications

(37 citation statements)

References 49 publications

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“…Phosphoryl transferase reactions with phosphorothioates (e.g., ATPγS) tend to be slower than the corresponding phosphoryl transfers and are chemistry-limited (38)(39)(40). Similarly, we have reported characteristics for a peptide substrate of the activated kinase with poor turnover but a fairly average Michaelis constant (27); the tetrafluorotyrosyl peptide F 4 -IRS727 (k cat ) 0.5 ( 0.1 s -1 , K M ) 0.21 ( 0.05 mM). Therefore, ATPγS and F 4 -IRS727 were used as controls for nonspecific effects of viscogens on the measurements of k cat .…”

Section: Methodsmentioning

confidence: 92%

“…Product inhibition studies were done with ADP over the range 50-200 µM, with 60 µM ATP and variable Y-IRS939 or 60 µM Y-IRS939 and variable ATP. Product inhibition by pY-IRS939 was over the range 600-1500 µM at fixed 80 µM ATP and variable IRS727, or 150 µM IRS727 and variable ATP; K M, IRS727 ) 180 ( 20 µM at pH 7.0 (27). The reactions with variable peptide concentrations were initiated with the addition of ATP, and those with variable ATP were initiated by the addition of peptide.…”

Section: Methodsmentioning

confidence: 99%

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Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Ablooglu

Kohanski

2000

Biochemistry

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The insulin receptor and many other protein kinases are activated by relief of intrasteric inhibition that is regulated by reversible phosphorylation. The changes accompanying activation of the insulin receptor's kinase domain were analyzed using steady-state kinetics, viscometric analysis, and equilibrium binding measurements. Peptide phosphorylation catalyzed by the unphosphorylated basal-state kinase is limited by a slow rate of the chemical step, and the activated enzyme is limited by product release rates. Underlying these changes were a 36-fold increase in the rate constant for the chemical step of the enzyme-catalyzed reaction, a 5-fold increase in the affinity for MgATP, and an 8-fold increase in the affinity for peptide substrate. This results in binding of substrates that is 2.2 kcal/mol more favorable and a free energy barrier for transition state formation that is lowered by 2.1 kcal/mol in the activated enzyme. Therefore, the change in conformational free energy inherent in the protein after autophosphorylation [Bishop, S. M., Ross, J. B. A., and Kohanski, R. A. (1999) Biochemistry 38, 3079-3089] is equally distributed between formation of the substrate ternary complex and formation of the transition state complex.

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Section: Methodsmentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Ablooglu

Kohanski

2000

Biochemistry

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“…The successful replacement of the Tyr phenol group with an anilino group, serving both as a hydrogen-bond donor and as part of the linker, was consistent with the known late proton removal from the hydroxy of the Tyr residue in the tyrosine PK reaction mechanism. [61] Also, a salt bridge between Lys 1030 and Glu 1047 was established by this crystal structure (PDB:1GAG), which is analogous to the salt bridge between Lys 72 and Glu 91 of PKAc, [32] the interaction required for efficient phosphoryl transfer.…”

Section: A C H T U N G T R E N N U N G Recognition Of Pksmentioning

confidence: 99%

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

2009

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Bisubstrate inhibitors consist of two conjugated fragments, each targeted to a different binding site of a bisubstrate enzyme. The design of bisubstrate inhibitors presupposes the formation of the ternary complex in the course of the catalyzed reaction. The principle advantage of bisubstrate inhibitors is their ability to generate more interactions with the target enzyme that could result in improved affinity and selectivity of the conjugates, when compared with single-site inhibitors. Among phosphotransferases, the approach was first successfully used for adenylate kinase in 1973. Since then, several types of bisubstrate inhibitors have been developed for protein kinases, including conjugates of peptides with nucleotides, adenosine derivatives and potent ATP-competitive inhibitors. Earlier bisubstrate inhibitors had pharmacokinetic qualities that were unsuitable for cellular experiments and hence were mostly used for in vitro studies. The recently constructed conjugates of adenosine derivatives and D-arginine-rich peptides (ARCs) possess high kinase affinity, high biological and chemical stability and good cell plasma membrane penetrative properties that enable their application in the regulation of cellular protein phosphorylation balances in cell and tissue experiments.

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“…In a mechanism-based approach to protein kinase inhibitors, bis-substrate compounds have been introduced in which the ␥-phosphate of ATP is covalently attached by a linker to the amino analogue of the OH acceptor amino acid located in a peptide of cognate substrate sequence (31)(32)(33). Noting that with the insulin receptor tyrosine kinase domain (IRK), the bissubstrate was not only an effective inhibitor but that, in the crystal structure, the peptide component was located with more order than in the complex of the non-covalently linked peptide with AMPPNP, we have explored the use bis-substrate compounds (hereafter referred to as bispeptides) in which the peptides are based on the model heptapeptide and the modified CDC6 peptide and in which the serine analogue amino-alanine is linked to ATP␥S via an acetyl bridge (Fig.…”

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confidence: 99%

The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition

Cheng

Noble

Skamnaki

et al. 2006

Journal of Biological Chemistry

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Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 Å from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the ␥-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower K m compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/ cyclin A activity against a heptapeptide substrate (K i ‫؍‬ 0.83 nM) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (K D 0.4 M) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.

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Probing the Catalytic Mechanism of the Insulin Receptor Kinase with a Tetrafluorotyrosine-containing Peptide Substrate

Cited by 32 publications

References 49 publications

Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition

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