Charbonneau H, Kumar S, Novack J P, Blumenthal D K, Griffin P R, Shabanowitz J, Hunt D F, Beavo J A, Walsh K A
Department of Biochemistry, University of Washington, Seattle 98195.
Biochemistry. 1991 Aug 13;30(32):7931-40. doi: 10.1021/bi00246a009.
The complete amino acid sequence of the 61-kDa calmodulin-dependent, cyclic nucleotide phosphodiesterase (CaM-PDE) from bovine brain has been determined. The native protein is a homodimer of N alpha-acetylated, 529-residue polypeptide chains, each of which has a calculated molecular weight of 60,755. The structural organization of this CaM-PDE has been investigated with use of limited proteolysis and synthetic peptide analogues. A site capable of interacting with CaM has been identified, and the position of the catalytic domain has been mapped. A fully active, CaM-independent fragment (Mr = 36,000), produced by limited tryptic cleavage in the absence of CaM, represents a functional catalytic domain. N-Terminal sequence and size indicate that this 36-kDa fragment is comprised of residues 136 to approximately 450 of the CaM-PDE. This catalytic domain encompasses a approximately 250 residue sequence that is conserved among PDE isozymes of diverse size, phylogeny, and function. CaM-PDE and its PDE homologues comprise a unique family of proteins, each having a catalytic domain that evolved from a common progenitor. A search of the sequence for potential CaM-binding sites revealed only one 15-residue segment with both a net positive charge and the ability to form an amphiphilic alpha-helix. Peptide analogues that include this amphiphilic segment were synthesized. Each was found to inhibit the CaM-dependent activation of the enzyme and to bind directly to CaM with high affinity in a calcium-dependent manner. This site is among the sequences cleaved from a 45-kDa chymotryptic fragment that has the complete catalytic domain but no longer binds CaM. These results indicate that residues located between position 23 and 41 of the native enzyme contribute significantly to the binding of CaM although the involvement of residues from additional sites is not excluded.
已确定来自牛脑的61 kDa钙调蛋白依赖性环核苷酸磷酸二酯酶（CaM-PDE）的完整氨基酸序列。天然蛋白质是Nα-乙酰化的529个残基多肽链的同源二聚体，每条链的计算分子量为60,755。利用有限蛋白酶解和合成肽类似物对该CaM-PDE的结构组织进行了研究。已鉴定出一个能够与钙调蛋白相互作用的位点，并绘制了催化结构域的位置。在没有钙调蛋白的情况下通过有限的胰蛋白酶切割产生的一个完全活性的、不依赖钙调蛋白的片段（Mr = 36,000）代表一个功能性催化结构域。N端序列和大小表明这个36 kDa的片段由CaM-PDE的136至约450位残基组成。这个催化结构域包含一个约250个残基的序列，该序列在不同大小、系统发育和功能的磷酸二酯酶同工酶中是保守的。CaM-PDE及其磷酸二酯酶同源物构成了一个独特的蛋白质家族，每个成员都有一个从共同祖先进化而来的催化结构域。对该序列中潜在的钙调蛋白结合位点进行搜索，仅发现一个具有净正电荷且能够形成两亲性α螺旋的15个残基片段。合成了包含该两亲性片段的肽类似物。发现每个类似物都能抑制该酶的钙调蛋白依赖性激活，并以钙依赖性方式与钙调蛋白直接高亲和力结合。该位点位于从一个45 kDa的胰凝乳蛋白酶片段切割下来的序列中，该片段具有完整的催化结构域但不再结合钙调蛋白。这些结果表明天然酶第23至41位之间的残基对钙调蛋白的结合有显著贡献，尽管不排除其他位点的残基也参与其中。
