STRUCTURE, FUNCTION, & GENETICS OF E COLI PHOSPHOTRIESTERASE HOMOLOG

抗体可以作为酶发挥作用，因为它们 拥有稳定的结构框架，可以支持多个 不同的结合位点突变。这允许对 各种各样的化学环境，包括那些 易受化学催化作用的。大自然已经选择了 Alpha/Beta-Barrel折叠作为一个类似的自适应框架 新酶的不同进化。约10%的已知 酶是α/β桶家族的成员，它们的 酶活性、催化效率和辅因子使用情况各不相同。 广泛地。磷酸三酯酶(Pte)是土壤中的一种a/b桶形酶。 催化水解物和解毒的细菌 磷三酯杀虫剂和神经毒剂。酶活性部位 含有两个锌原子，通过他的侧链与蛋白质结合 由氨甲酰化的赖氨酸侧链连接。这种酶显著地 高效；磷酸三酯酶的催化速率仅受 酶和底物在溶液中的扩散速率。因为 该酶的磷酸三酯底物是合成化合物。 在没有已知的生物对应物的情况下，考虑这一点很有趣 导致PTE发明的进化路径--什么是 这项新活动的起点和方式是什么？vbl.使用 同源搜索技术，我们发现了一个开放的大肠杆菌基因组 与磷酸三酯酶高度相似的阅读框架(ORF)。 这个ORF位于大约74分钟处。在大肠杆菌的染色体上 一组新发现的似乎编码所用酶的ORF 在有机磷新陈代谢中。我们已将该大肠杆菌同源基因命名为 磷酸三酯酶同源蛋白(PHP)。多肽序列 与PTE和四个His残基的同源性为28% PTE中锌原子的配位体在PHP中是保守的。此外，PHP 的相应位置包含谷氨酸残基 Pte的氨甲酰化赖氨酸。基于这种结构同源性，我们 可以预见：1)PHP有一个a/b桶结构；2)PHP是一个 酶；3)PHP是一种金属酶；以及4)PHP是 PTE起源的酶亚家族。两国之间的关系 PTE和PHP为研究路径提供了一个独特的研究机会 在酶的进化中。PHP代表起点，PTE代表终点 点，问题是我们是否能重建 实验室中将PHP转化为PTE的突变。我们也是 对了解PHP的自然生物学功能感兴趣，因为 这可能为为什么PHP子家族成员被 土壤细菌成为一种新的磷酸三酯水解酶。

Antibodies can be made to function as enzymes bec ause they possess a stable structural framework that can support a number of different combining site mutations. This allows for sampling of a wide variety of chemical environments, including those that are amenable to chemical catalysis. Nature has selected the alpha/beta-barrel fold as a similar adaptable framework for the divergent evolution of new enzymes. Approximately 10% of all known enzymes are members of the alpha/beta barrel family and their enzymatic activities, catalytic efficiencies, and cofactor usage vary widely. Phosphotriesterase (PTE) is an a/b barrel enzyme from soil bacteria which catalyzes the hydrolysis and detoxification of phosphotriester insecticides and nerve agents. The enzyme active site contains two zinc atoms bound to the protein with His side chains and bridged by a carbamoylated Lys sidechain. The enzyme is remarkably efficient; the catalytic rate of phosphotriesterase is limited only by the diffusion rate of enzyme and substrate in solution. Because the phosphotriester substrates for this enzyme are synthetic compounds with no known biological counterpart, it is interesting to consider the evolutionary pathway that led to the invention of PTE--what was the starting point and how was this new activity created? Using homology searching techniques, we have found an E. coli genomic open reading frame (ORF) that is highly similar to phosphotriesterase. This ORF is located at about 74 min. on the E. coli chromosome in a cluster of newly identified ORF's that appear to encode enzymes used in organophosphate metabolism. We have named the E. coli homolog phosphotriesterase homology protein (PHP). The polypeptide sequence of PHP is 28% identical to PTE and the four His residues that are ligands to the Zn atoms in PTE are conserved in PHP. In addition, PHP contains a Glu residue at the corresponding position of the carbamoylated Lys of PTE. Based on this structural homoloy, we predict that: 1) PHP has an a/b barrel structure; 2) PHP is an enzyme; 3) PHP is a metallo enzyme; and 4) PHP is a member of the enzyme subfamily from which PTE originated. The relationship between PTE and PHP presents a unique research opportunity to study a pathway in enzyme evolution. PHP represents the start point and PTE the end point, and the question is whether we can recreate the mutations in the laboratory that convert PHP into PTE. We are also interested in understanding the natural biological function of PHP, as this may provide clues to why a PHP subfamily member was recruited by soil bacteria to become a new phosphotriester hydrolase.