Influence of key residues on the reaction mechanism of the cAMP-dependent protein kinase

Citation
Mc. Hutter et V. Helms, Influence of key residues on the reaction mechanism of the cAMP-dependent protein kinase, PROTEIN SCI, 8(12), 1999, pp. 2728-2733
Citations number
25
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
09618368 → ACNP
Volume
8
Issue
12
Year of publication
1999
Pages
2728 - 2733
Database
ISI
SICI code
0961-8368(199912)8:12<2728:IOKROT>2.0.ZU;2-0
Abstract
The reaction mechanism of the catalytic phosphoryl transfer of cAMP-depende nt protein kinase (cAPK) was investigated by semi-empirical AM1 molecular o rbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier i s calculated as 20.7 kcal mol(-1) and the reaction itself to be exothermic by 12.2 kcal mol(-1). The active site residue Asp166, which was often propo sed to act as a catalytic base, does not accept a proton in any of the reac tion steps. Instead, the hydroxyl hydrogen of serine is shifted to the simu ltaneously transferred phosphate group of ATP. Although the calculated tran sition state geometry indicates an associative phosphoryl transfer, no conc entration of negative charge is found. To study the influence of protein mu tations on the reaction mechanism, we compared two-dimensional energy hyper surfaces of the protein kinase wild-type model and a corresponding mutant i n which Asp166 was replaced by alanine. Surprisingly, they show similar ene rgy profiles despite the experimentally known decrease of catalytic activit y for corresponding mutants. Furthermore, a model structure was examined, w here the charged NH3 group of Lys168 was replaced by a neutral methyl group . The energetic hypersurface of this hypothetical mutant shows two possible pathways for phosphoryl transfer, which both require significantly higher activation energies than the other systems investigated, while the energeti c stabilization of the reaction product is similar in all systems. As the p osition of the amino acid side chains and the substrate peptide is virtuall y unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer its elf, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys168, however, is necessary to stabi lize the intermediate reaction states, particularly the side chain of the s ubstrate peptide.