Comparative studies on structures of S-adenosylmethionine binding sites of mammalian methyltransferases

哺乳动物甲基转移酶S-腺苷甲硫氨酸结合位点结构的比较研究

• 批准号: 05680522
• 负责人: GOMI Tomoharu
• 金额: $ 1.28万
• 依托单位: TOYAMA MEDICAL AND PHARMA CEUTICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680522/
• 关键词: Methyltransferase; S-Adenosylmethionine; Guanidinoacetate methyltransferase; Glycine methyltransferase; Affinity labeling; Site-directed mutagenesis; Positive cooperativity; Amino-terminal acetylation

1. Guanidinoacetate methyltransferase (GAMT) : (1) Tyr-136 that is photoaffinity -labeled by AdoMet resides in a region whose structural feature is shared by most mammalian methyltransferases (mMTs). Amino acid replacements were introduced to the region. The results of precise kinetic analyzes of mutant enzymes indicate that Asp-134 is crucial for binding AdoMet. (2) We found that mMTs share a sequence motif similar to one that is common in nucleotide-binding proteins. Studies by site-directed mutagenesis suggested the importance of the motif for the enzymatic activity of GAMT.2. Glycine methyltransferase (GMT) : (1) It is reported that rat GMT shows positive cooperativity toward AdoMet while rabbit enzyme dose not. Cloning and sequencing of GMTs from rabbit, human, and pig livers charified that all GMTs including rat enzyme have very similar structures, and kinetic analyzes with liver extracts revealed that they all exhibit the cooperativity toward AdoMet. (2) The recombinant rabbit GMT did not show the kinetic cooperativity. The only structural difference between recombinant-and liver enzyme was that the amino-terminal Val residue of the former is free while that of the latter is acetylated.This cbservation and the result of pH study suggest that the acetylation confers on GMT the cooperativity toward AdoMet by masking amino-terminal positive charge. (3) Recombinant rat GMT was crystallized and preliminary X-ray diffraction data set was obtained.

1.乙酸胍甲基转移酶(GAMT)：(1)由ADOMet光亲和标记的Tyr-136位于大多数哺乳动物甲基转移酶(MMT)所共有的结构特征区域。氨基酸替代品被引入该地区。突变酶的精确动力学分析结果表明，Asp-134是结合ADOMet的关键酶。(2)我们发现MMT具有与核苷酸结合蛋白相似的序列基序。通过定点突变的研究表明该基序对于GAMT的酶活性具有重要意义。甘氨酸甲基转移酶(GMT)：(1)据报道，大鼠GMT对ADO-Met具有正协同作用，而兔酶则不具有正协同作用。兔、人和猪肝脏GMT的克隆和测序表明，包括大鼠酶在内的所有GMT具有非常相似的结构，与肝脏提取物的动力学分析表明，它们都对Adobe Met表现出协同作用。(2)重组兔GMT不表现出动力学协同作用。重组酶和肝酶唯一的结构差异是前者的氨基端Val残基是游离的，而后者是乙酰化的。这一观察结果和pH研究结果表明，乙酰化通过掩蔽氨基端的正电荷在GMT上赋予了对Adobe Met的协同性。(3)对重组大鼠GMT进行晶化，得到了初步的X射线衍射数据集。

项目成果

Ogawa et al.: "Mammalian glycinc N-mcthyltransfcrsc.Comparative kinctic and Structural Properties of the cnzymes from human,rat,rabbit and pig livers" Comparative Biochemistry and Physiology. 106B. 601-611 (1993)

Okawa 等人：“哺乳动物甘氨酸 N-甲基转移酶。来自人、大鼠、兔和猪肝脏的酶的比较运动和结构特性”比较生物化学和生理学。

Takata et al.: "Rat guanidinoacetate methyltransferase.Effect of site-directed alteration of an aspartic residue that is conserved across most mammalian S-adenosylmethionine-dependent methyltransferases." Journal of Biological Chemistry. 269. 5537-5542 (1

Takata 等人：“大鼠胍基乙酸甲基转移酶。对大多数哺乳动物 S-腺苷甲硫氨酸依赖性甲基转移酶中保守的天冬氨酸残基进行定点改变的影响。”

Takata et al.: "Mammalian glycine N-methyltransferase. Comparative kinetic and structural properties of the enzymes from human, rat, rabbit and pig livers." J.Biol.Chem.269 (6). 4084-4091 (1994)

Takata 等人：“哺乳动物甘氨酸 N-甲基转移酶。人、大鼠、兔和猪肝脏酶的动力学和结构特性比较。”

Ogawa et al.: "Mammalian glycine N-methyltransferase. Comparative kinetic and structural properties of the enzymes from human, rat, rabbit and pig livers." Comp.Biochem.Physiol.-B : Comp.Biochem.106 (3). 601-611 (1993)

Okawa 等人：“哺乳动物甘氨酸 N-甲基转移酶。人、大鼠、兔和猪肝脏酶的动力学和结构特性比较。”

Aksamit et al.: "The role of cysteine 78 in fluorosulfonylbenzoyladenosine inactivation of rat liver S-adenosylhomocysteine hydrolase." J.Biol.Chem.269 (8). 5537-5542 (1994)

Aksamit 等人：“半胱氨酸 78 在氟磺酰苯甲酰腺苷灭活大鼠肝脏 S-腺苷高半胱氨酸水解酶中的作用。”