Myllyharju J, Kivirikko K I
Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu, Finland.
EMBO J. 1999 Jan 15;18(2):306-12. doi: 10.1093/emboj/18.2.306.
Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase.
collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase
脯氨酰4-羟化酶（EC 1.14.11.2）催化-X-Pro-Gly-序列的羟化反应，在所有胶原蛋白的合成中起核心作用。[α(I)]2β2型I酶可被聚（L-脯氨酸）有效抑制，而[α(II)]2β2型II酶则不能。我们在此报告，脯氨酰4-羟化酶的聚（L-脯氨酸）和（Pro-Pro-Gly）10肽底物结合结构域与催化结构域不同，由约100个氨基酸组成。在对人I型酶四聚体进行有限蛋白酶解后，从517个氨基酸的α(I)亚基中约140位残基开始的10 - 19 kDa肽段仍与聚（L-脯氨酸）琼脂糖结合。在大肠杆菌中表达的与α(I)亚基138 - 244位残基相对应的重组多肽是可溶的，能有效结合到聚（L-脯氨酸）琼脂糖上，并可用（Pro-Pro-Gly）10洗脱。该多肽不同于SH3和WW结构域以及脯肌动蛋白，因此代表了一种新型的富含脯氨酸的肽结合模块。对含有突变α亚基的酶四聚体的研究表明，α(II)亚基中对应于α(I)亚基中Ile182和Tyr233位置的谷氨酸和谷氨酰胺的存在，解释了II型脯氨酰4-羟化酶缺乏聚（L-脯氨酸）结合的大部分原因。
胶原蛋白/双加氧酶/肽结合结构域/富含脯氨酸/脯氨酰羟化酶
