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
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.