2.8-ANGSTROM STRUCTURE OF YEAST SERINE CARBOXYPEPTIDASE
2.8-ANGSTROM STRUCTURE OF YEAST SERINE CARBOXYPEPTIDASE
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DOI：
10.1021/bi00203a007
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发表时间：
1994-09-20
期刊：
影响因子：
2.9
通讯作者：
REMINGTON, SJ
中科院分区：
文献类型：
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作者：
ENDRIZZI, JA;BREDDAM, K;REMINGTON, SJ
采用多个同晶置换和晶体学精修的方法，对一种新的去糖基化方法得到的酿酒酵母丝氨酸羧肽酶（CPD-Y）的结构进行了测定。该模型包含3333个非氢原子，所有421个氨基酸，4个碳水化合物残基中的3个，5个二硫键和38个水分子。在20.0和2.8埃分辨率之间观察到的10909次反射的标准晶体学R因子为0.162。该模型的键长和键角与理想值的均方根偏差分别为0.016埃和2.7度，与受约束的热参数的均方根偏差为7.9埃（2）。CPD-Y，表现出对疏水肽的偏好，与二聚小麦丝氨酸羧肽酶II（CPD-WII）有较远的关系，后者对碱性肽有偏好。两种结构的比较表明，在结合位点中CPD-Y中的疏水残基取代CPD-WII中的带负电荷的残基是造成这种差异的主要原因。催化残基在两个分子中的构型基本相同，包括三个活性位点残基（Ser 146，Gly 52和Gly 53）的应变主链构象角以及Glu 145和Glu 65的羧基之间的不寻常氢键。抑制剂苄基琥珀酸的结合表明肽底物的C-末端羧酸结合位点是Asn 51、Gly 52、Glu 145和His 397，并且“含氧阴离子空穴”由Gly 53和Tyr 147的酰胺组成。该研究的一个令人惊讶的结果是，由残基180-317组成的结构域在两种分子中的结构非常不同，这些结构域在活性位点周围的高度保守的核心中形成了很大程度上的α-螺旋插入。有人建议，这些领域的发展速度比其他部分的分子，并参与底物识别。
The structure of monomeric serine carboxypeptidase from Saccharomyces cerevisiae (CPD-Y), deglycosylated by an efficient new procedure, has been determined by multiple isomorphous replacement and crystallographic refinement. The model contains 3333 non-hydrogen atoms, all 421 amino acids, 3 of 4 carbohydrate residues, 5 disulfide bridges, and 38 water molecules. The standard crystallographic R-factor is 0.162 for 10 909 reflections observed between 20.0- and 2.8-Angstrom resolution. The model has rms deviations from ideality of 0.016 Angstrom for bond lengths and 2.7 degrees for bond angles and from restrained thermal parameters of 7.9 Angstrom(2). CPD-Y, which exhibits a preference for hydrophobic peptides, is distantly related to dimeric wheat serine carboxypeptidase II (CPD-WII), which has a preference for basic peptides. Comparison of the two structures suggests that substitution of hydrophobic residues in CPD-Y for negatively charged residues in CPD-WII in the binding site is largely responsible for this difference. Catalytic residues are in essentially identical configurations in the two molecules, including strained main-chain conformational angles for three active site residues (Ser 146, Gly 52, and Gly 53) and an unusual hydrogen bond between the carboxyl groups of Glu 145 and Glu 65. The binding of an inhibitor, benzylsuccinic acid, suggests that the C-terminal carboxylate binding site for peptide substrates is Asn 51, Gly 52, Glu 145, and His 397 and that the ''oxyanion hole'' consists of the amides of Gly 53 and Tyr 147. A surprising result of the study is that the domains consisting of residues 180-317, which form a largely alpha-helical insertion into the highly conserved cores surrounding the active site, are quite different structurally in the two molecules. It is suggested that these domains have evolved much more rapidly than other parts of the molecule and are involved in substrate recognition.