Unveiling the substrate specificity of meprin beta on the basis of the site in protein kinase A cleaved by the kinase splitting membranal proteinase.
The kinase splitting membranal proteinase (KSMP) is a metalloendopeptidase that inactivates the catalytic (C) subunit of protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at Glu332-Glu333, within the cluster of acidic amino acids (Asp328-Glu334) of the kinase. The Km values of KSMP and of meprin beta (which reproduces KSMP activity) for the C-subunit are below 1 microM. The Km for peptides containing a stretch of four Glu residues are in the micromolar range, illustrating the significant contribution of this cluster to the substrate recognition of meprin beta. This conclusion is supported by a systematic study using a series of the C-subunit mutants with deletions and mutations in the cluster of acidics. Hydrophobic amino acids vicinal to the cleavage site increase the Kcat of the proteinase. These studies unveil a new specificity for meprin beta, suggesting new substrates that are 1-2 orders of magnitude better in their Km and Kcat than those commonly used for meprin assay. A search for substrates having such a cluster of acidics and hydrophobics, which are accessible to meprin under physiological conditions, point at gastrin as a potential target. Indeed, meprin beta is shown to cleave gastrin at its cluster of five glutamic acid residues and also at the M-D bond within its WMDF-NH2 sequence, which is indispensable for all the known biological activities of gastrins. The latter meprin cleavage will lead to the inactivation of gastrin and thus to the control of its activity.
Chestukhin
A
A
Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, 76100, Israel.
Litovchick
L
L
Muradov
K
K
Batkin
M
M
Shaltiel
S
S
D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D016415:Sequence Alignment; D017384:Sequence Deletion; D013379:Substrate Specificity
D017868:Cyclic AMP-Dependent Protein Kinases; D010450:Endopeptidases; D008666:Metalloendopeptidases; C095663:meprin B; C059784:kinase-splitting membranal proteinase