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QBIC'99: Mechanism of Phosphoryltransfer of cAMP-Dependent Protein Kinase

 

Mechanism of Phosphoryltransfer of cAMP-Dependent Protein Kinase Studied by Quantum-Chemical Calculations

M. C. Hutter and V. Helms

Max-Planck Insitute for Biophysics, Frankfurt, Germany

Abstract

cAMP-dependent protein kinase phosphorylates Ser- and Thr-residues of a variety of peptides. The protein is extremely well characterized biochemically, biophysically, and structurally. For example, crystallographic structures of substrate and product states were determined by Susan Taylor and co-workers and the reaction mechanism was studied by stopped-flow fluorescence. However many of the mechanistic details are still unresolved. We investigated the catalytic step by AM1 calculations on a subsystem of the active site that includes 125 atoms. The hydroxyl proton of the accepting Ser-residue is very likely accepted by the phosphate group itself rather than by nearby Asp166, in agreement with recent computational results by Ian Hillier and others. The activation barrier for the phosphoryl-transfer was calculated as ca. 20-25 kcal/mol for wild-type enzyme and two Asp166 mutants. Similar energies were also obtained for mutating Lys168 into a neutral isoform. Our work clarifies that the characteristic DLK motif of protein kinases is not essential for the catalytic step, but must have structural function or may be involved in correct positioning of reactant and product.
 
 

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