Catalytic Mechanism of Guanidinoacetate Methyltransferase: Crystal Structures of Guanidinoacetate Methyltransferase Ternary Complexes
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- 作者：Junichi Komoto ; Taro Yamada ; Yoshimi Takata ; Kiyoshi Konishi ; Hirofumi Ogawa ; Tomoharu Gomi ; Motoji Fujioka ; and Fusao Takusagawa
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：November 16, 2004
- 年：2004
- 卷：43
- 期：45
- 页码：14385 - 14394
- 全文大小：841K
- 年卷期：v.43,no.45(November 16, 2004)
- ISSN：1520-4995
文摘
Guanidinoacetate methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatinebiosynthesis. The enzyme is found in abundance in the livers of all vertebrates. The intact GAMT fromrecombinant rat liver has been crystallized with an inhibitor S-adenosylhomocysteine (SAH) and a substrateguanidinoacetate (GAA), and with SAH and an inhibitor guanidine (GUN). These ternary complex structureshave been determined at 2.0 Å resolution. GAMT has an 
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open-sandwich structure, and the N-terminalsection (residues 1-42) covers the active site entrance so that the active site is not visible. SAH hasextensive interactions with GAMT through H-bonds and hydrophobic interactions. The guanidino groupsof GAA and GUN form two pairs of H-bonds with E45 and D134, respectively. The carboxylate groupof GAA interacts with the backbone amide groups of L170 and T171. A model structure of GAMTcontaining the two substrates (SAM and GAA) was built by attaching a methyl group (CE) on SD of thebound SAH. On the basis of this model structure, a catalytic mechanism of GAMT is proposed. Theactive site entrance is opened when the N-terminal section is moved out. GAA and SAM enter the activesite and interact with the amino acid residues on the surface of the active site by polar and nonpolarinteractions. OD1 of D134 and CE of SAM approach NE of GAA from the tetrahedral directions. TheOD1···NE and CE···NE distances are 2.9 and 2.2 Å, respectively. It is proposed that three slightly negativelycharged carbonyl oxygen atoms (O of T135, O of C168, and OB of GAA) around OD1 of D134 increasethe pKa of OD1 so that OD1 abstracts the proton on NE. A strong nucleophile is generated on the deprotonatedNE of GAA, which abstracts the methyl group (CE) from the positively charged SD of SAM, and creatine(methyl-GAA) and SAH (demethyl-SAM) are produced. E45, D134, and Y221 mutagenesis studies supportthe proposed mechanism. A mutagenesis study and the inhibitory mechanism of guanidine analoguessupport the proposed mechanism.
/ open-sandwich structure, and the N-terminalsection (residues 1-42) covers the active site entrance so that the active site is not visible. SAH hasextensive interactions with GAMT through H-bonds and hydrophobic interactions. The guanidino groupsof GAA and GUN form two pairs of H-bonds with E45 and D134, respectively. The carboxylate groupof GAA interacts with the backbone amide groups of L170 and T171. A model structure of GAMTcontaining the two substrates (SAM and GAA) was built by attaching a methyl group (CE) on SD of thebound SAH. On the basis of this model structure, a catalytic mechanism of GAMT is proposed. Theactive site entrance is opened when the N-terminal section is moved out. GAA and SAM enter the activesite and interact with the amino acid residues on the surface of the active site by polar and nonpolarinteractions. OD1 of D134 and CE of SAM approach NE of GAA from the tetrahedral directions. TheOD1···NE and CE···NE distances are 2.9 and 2.2 Å, respectively. It is proposed that three slightly negativelycharged carbonyl oxygen atoms (O of T135, O of C168, and OB of GAA) around OD1 of D134 increasethe pKa of OD1 so that OD1 abstracts the proton on NE. A strong nucleophile is generated on the deprotonatedNE of GAA, which abstracts the methyl group (CE) from the positively charged SD of SAM, and creatine(methyl-GAA) and SAH (demethyl-SAM) are produced. E45, D134, and Y221 mutagenesis studies supportthe proposed mechanism. A mutagenesis study and the inhibitory mechanism of guanidine analoguessupport the proposed mechanism.