Chestukhin A, Litovchick L, Muradov K, Batkin M, Shaltiel S
Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, 76100, Israel.
J Biol Chem. 1997 Feb 7;272(6):3153-60. doi: 10.1074/jbc.272.6.3153.
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.
激酶裂解膜蛋白酶（KSMP）是一种金属内肽酶，它通过切除蛋白激酶A（PKA）催化（C）亚基的羧基末端尾巴来使其失活。在此我们表明，这种切割发生在激酶酸性氨基酸簇（Asp328 - Glu334）内的Glu332 - Glu333处。KSMP和meprin β（重现KSMP活性）对C亚基的Km值低于1微摩尔。对于含有一段四个Glu残基的肽段，其Km值在微摩尔范围内，这说明该簇对meprin β的底物识别有重要贡献。这一结论得到了一系列使用在酸性氨基酸簇中有缺失和突变的C亚基突变体的系统研究的支持。切割位点附近的疏水氨基酸增加了蛋白酶的催化常数（Kcat）。这些研究揭示了meprin β的一种新特异性，表明存在一些新底物，其Km和Kcat比常用于meprin检测的底物要好1 - 2个数量级。寻找在生理条件下meprin可接近的具有这种酸性和疏水氨基酸簇的底物，发现胃泌素是一个潜在靶点。实际上，已表明meprin β在胃泌素的五个谷氨酸残基簇处以及在其WMDF - NH2序列内对所有已知胃泌素生物活性都不可或缺的M - D键处切割胃泌素。meprin的后一种切割将导致胃泌素失活，从而对其活性进行调控。
