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Volume 272, Number 6, Issue of February 7, 1997 pp. 3153-3160
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Unveiling the Substrate Specificity of Meprin  on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

(Received for publication, September 30, 1996)

From the Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, 76100, Israel

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 K_m values of KSMP and of meprin  (which reproduces KSMP activity) for the C-subunit are below 1 µM. The K_m 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 . 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 K_cat of the proteinase. These studies unveil a new specificity for meprin , suggesting new substrates that are 1-2 orders of magnitude better in their K_m and K_cat 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  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.

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