Journal of Biological Chemistry
Volume 276, Issue 50, 14 December 2001, Pages 46933-46940
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ENZYME CATALYSIS AND REGULATION
Multiple Activation Loop Conformations and Their Regulatory Properties in the Insulin Receptor's Kinase Domain*

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Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q ≈ D1132N ≥ D1161A > Y1162F ≈ K1085N > Q1208S ≥ wild-type. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple off-versus-on switch for kinase activation.

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Published, JBC Papers in Press, October 10, 2001, DOI 10.1074/jbc.M107236200

*

This work was supported by Grant DK59522 from the National Institutes of Health (to R. A. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Carnegie Institution of Washington, Baltimore, MD 21210.

§

Current address: Dept. of Chemistry, University of Montana, Missoula, MT 59812.