Catalysis of proline-directed protein phosphorylation by peptidyl-prolyl cis/trans isomerases.
Proline-directed protein phosphorylation was shown to depend on the capacity of the targeted Ser(Thr)-Pro bond to exhibit conformational polymorphism. The cis/trans isomer specificity underlying ERK2-catalyzed phosphate transfer leads to a complete discrimination of the cis Ser(Thr)-Pro conformer of oligopeptide substrates. We investigated in vitro the ERK2-catalyzed phosphorylation of Aspergillus oryzae RNase T1 containing two Ser-Pro bonds both of which share high stabilization energy in their respective native state conformation, the cis Ser54-Pro and the trans Ser72-Pro moiety. Despite trans isomer specificity of ERK2, a doubly phosphorylated RNase T1 was found as the final reaction product. Similarly, the RNase T1 S54G/P55N and RNase T1 P73V variants, which retain the prolyl bond conformations of the RNase T1-wt, were both monophosphorylated with a catalytic efficiency kcat/KM of 425 M(-1) s(-1) and 1228 M(-1) s(-1), respectively. However, initial phosphorylation rates did not depend linearly on the ERK2 concentration. The phosphorylation rate of the resulting plateau region at high ERK2 concentrations can be increased up to threefold for the RNase T1 P73V variant in the presence of the peptidyl-prolyl cis/trans isomerase Cyclophilin 18, indicating a conformational interconversion as the rate limiting step in the catalyzed phosphate group transfer. Using peptidyl-prolyl cis/trans isomerases with different substrate specificity, we identified a native state conformational equilibrium of the Ser54-Pro bond with the minor trans Ser54-Pro bond as the phosphorylation-sensitive moiety. This technique can therefore be used for a determination of the ratio and the interconversion rates of prolyl bond isomers in the native state of proteins.
Weiwad
Matthias
M
Research Unit Enzymology of Protein Folding, Max-Planck Society, Weinbergweg 22, D-06120 Halle/Saale, Germany.
Werner
Andreas
A
Rücknagel
Peter
P
Schierhorn
Angelika
A
Küllertz
Gerd
G
Fischer
Gunter
G
D001236:Aspergillus oryzae / Q000201:enzymology; D002384:Catalysis; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000378:metabolism*; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism*
D011392:Proline; D019950:Mitogen-Activated Protein Kinase 1; D006163:Ribonuclease T1; D019696:Peptidylprolyl Isomerase