Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase.
The effect of prolyl bond isomers on the substrate recognition capabilities of various endoproteases may be investigated in a reaction where both cis/trans isomers co-exist. Here we address the question of whether enzyme reactions at the side chain of an amino acid preceding proline proceed through an isomer specific pathway. The proline-directed p42 mitogen-activated protein kinase (ERK2) was used to phosphorylate the serine side chain in Pro-Arg-Ser-Pro-Phe-4-nitroanilide under conditions where different amounts of cis prolyl isomer of the substrate were present. Initial phosphorylation rates were calculated ranging between zero at 100% cis isomer and around 60 pM/min at the equilibrium content of 83.5% trans isomer. In the presence of the peptidyl-prolyl cis/trans isomerase human hFKBP12 (500 nM), cis/trans isomerization proceeds rapidly, permitting the maximal phosphorylation rate to be observed in the dead time of the experiment. Results show that correct signature sequences are not sufficient to render potential substrates reactive to proline-directed enzymatic phosphorylations, but that the conformational state of the peptide bond following serine (threonine) is a critical determinant. Therefore, catalysis by peptidyl-prolyl cis/trans isomerases may add a new level of control to intracellular protein phosphorylations.
Weiwad
M
M
Max-Planck Research Unit Enzymology of Protein Folding, Weinbergweg 22, D-06120, Halle, Germany.
Küllertz
G
G
Schutkowski
M
M
Fischer
G
G
D020816:Amino Acid Motifs; D000818:Animals; D016384:Consensus Sequence; D006801:Humans; D020104:Immunophilins / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D013379:Substrate Specificity; D022021:Tacrolimus Binding Proteins; D013816:Thermodynamics
D010455:Peptides; D019950:Mitogen-Activated Protein Kinase 1; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase