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 reac tions at the side chain of an amino acid preceding proline proceed through an isomer specific pathway, The proline-directed p42 mitogen-activated prot ein kinase (ERK2) was used to phosphorylate the serine side chain in Pro-Ar g-Ser-Pro-Phe-4-nitroanilide under conditions where different amounts of ci s prolyl isomer of the substrate were present. Initial phosphorylation rate s 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 th e peptidyl-prolyl cis/trans isomerase human hFKBP12 (500 nM), cis/trans iso merization 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 react ive to proline-directed enzymatic phosphorylations, hut that the conformati onal state of the peptide bond following serine (threonine) is a critical d eterminant. Therefore, catalysis by peptidyl-prolyl cis/trans isomerases ma y add a new level of control to intracellular protein phosphorylations, (C) 2000 Federation of European Biochemical Societies. Published by Elsevier S cience B.V. All rights reserved.