Siebert Xavier, Eipper Betty A, Mains Richard E, Prigge Sean T, Blackburn Ninian J, Amzel L Mario
Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Biophys J. 2005 Nov;89(5):3312-9. doi: 10.1529/biophysj.105.066100. Epub 2005 Aug 12.
Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met(314) and two His residues as ligands. Mutation of Met(314) to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met(314) as a CuM ligand, we determined the structure of the Met(314)Ile-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The Met(314)Ile-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the Met(314)Ile-PHM crystal form and showed that it does not differ from the structure of wild-type (wt)-PHM in the original crystals. Met(314)Ile-PHM was also shown to be less stable than wt-PHM by differential scanning calorimetry. Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.
许多生物活性肽需要其羧基末端酰胺化才能展现出完全的生物活性。肽基甘氨酸α-羟化单加氧酶（PHM；EC 1.14.17.3）催化该反应两个步骤中的第一步，它由两个结构域组成，每个结构域结合一个铜原子（CuH和CuM）。CuM位点包括Met(314)和两个组氨酸残基作为配体。将Met(314)突变为异亮氨酸会使PHM失活，但对氧化型酶的扩展X射线吸收精细结构（EXAFS）光谱影响极小，这意味着它对CuM位点的稳定性贡献很小。为了表征Met(314)作为CuM配体的作用，我们测定了Met(314)Ile-PHM突变体的结构。由于突变蛋白在原始野生型蛋白的条件下无法结晶，因此该结构测定需要找到一种新的晶体形式。Met(314)Ile-PHM突变体结构证实该突变不会消除CuM与酶的结合，但会引起其他结构扰动，从而影响酶的整体稳定性和CuH位点的完整性。为了消除晶体接触的可能影响，我们重新测定了处于Met(314)Ile-PHM晶体形式的野生型PHM的结构，并表明它与原始晶体中野生型（wt）-PHM的结构没有差异。差示扫描量热法也表明Met(314)Ile-PHM比野生型PHM稳定性更低。结构和量热研究均表明，除了已确定的催化作用外，CuM位点还具有结构作用。
