鳥胺酸胺基異構酶之性質探討; Properties of Adenosylcobalamin-dependent D-Ornithine Aminomutase

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Title:	鳥胺酸胺基異構酶之性質探討;Properties of Adenosylcobalamin-dependent D-Ornithine Aminomutase
Authors:	林麗瑩;LiYing Lin
Contributors:	中國醫藥學院醫學研究所
Keywords:	鳥胺酸;胺基異構;輔;B12;Ornithine;Aminomutase;Adenosylcobalamin
Date:	1992
Issue Date:	2009-12-23
Abstract:	Clostridium sticklandii菌株內之D-ornithine aminomutase是以四聚體的型式存在，由兩種次單元組成：E2S2，E subunit之分子量為82900 Da，而S subunit分子量為12800 Da。D-ornithine aminomutase需要結合輔酶B6 ( pyridoxal phosphate，PLP )及輔酶B12 (adenosyl-cobalamin，AdoCbl)來參與它催化D-ornithine與2,4-diaminopentanoic acid之間結構轉變的反應；酵素反應平衡常數的測量結果為0.68及0.85，結果顯示D-ornithine aminomutase傾向催化2,4-diaminopentanoic acid產生D-ornithine。酵素活性反應的測量結果得知2,4-diamino-n-butyric acid可以抑制酵素活性，是為競爭型抑制劑，對D-ornithine aminomutase而言，2,4-diamino-n-butyric acid的Ki = 96.4 μM，化合物結構中的兩個胺基及羰基與酵素之間的互動有密切的關係；使用核磁共振光譜檢查酵素反應產物，除了D-ornithine之外，2,4-diamino-n-butyric acid、2,5-diaminopentanol與1,4-diaminobutane這些化合物，都不會被D-ornithine aminomutase催化成產物，顯示D-ornithine aminomutase對受質的專一性很高。調查D-ornithine、2,4-diamino-pentanoic acid、2,4-diamino-n-butyric acid、4-aminopentanoic acid、1,3-diamino- propane、1,4-diaminobutane與PLP形成的schiff base的光譜圖，結果不同於與enzyme-bound PLP形成的光譜。利用毛細管電泳可以得知蛋白質的流體動力半徑及有效電荷數，S次單元半徑為2.03 nm，PLP及AdoCbl-bound D-ornithine aminomutase半徑為5.86、5.17 nm；在pH=7時，PLP-bound D-ornithine aminomutase的有效電荷數為25.75個負電荷，AdoCbl-bound D-ornithine aminomutase的有效電荷數為26.73個負電荷。; D-Ornithine aminomutase comprises two strongly associating subunit, S and E, with molecular masses of 12,800 Da and 82,900 Da, respectively. The apoenzyme combines with adenosylcobalamin and pyridoxal phosphate to form the active holoenzyme. It catalyzes the reversible interconversion of D-ornithine to (2R,4S)-2,4-diaminopentanoic acid. The equilibrium direction in this reaction is in favor of the less branched D-ornithine with a Keq of 0.68. The substrate analogs, 2,4-diamino-n-butyric acid, 2,5-diaminopentanol, 1,3- diaminopropane, 1,4-diamino-butane, and 4-aminopentanoic acid, were studied kinetically; only 2,4-diamino-n-butyric acid behaved as a competitive inhibitor of D-ornithine aminomutase with a Ki of 96.4 μM. The carboxyl group and amino groups of D-ornithine and 2,4-diaminopentanoic acid play important roles in the catalytic mechanism of D-ornithine aminomutase that of the UV-visible spectrum of pyridoxal phosphate is different from D-ornithine aminomutase. In addition, capillary electrophoresis was used to measure the effective charge and size of the protein. The results show that the effective charge of the enzyme is negative. The radiuses of S subunit, pyridoxal phosphate-bound D-ornithine aminomutase, and adenosylcobalamin-bound D-ornithine aminomutase are 2.03, 5.86, and 5.17 nm, respectively.
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