Shenoy B C, Wood H G
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
FASEB J. 1988 May;2(8):2396-401. doi: 10.1096/fasebj.2.8.3360240.
The synthetase that attaches biotin to the aposubunit of transcarboxylase (biotin-[methylmalonyl-CoA-carboxyltransferase]ligase) (EC 6.3.4.9) was purified to homogeneity by ion-exchange chromatography on cellulose DE-52 and CM-cellulose. The synthetase is a monomer of molecular weight 30,000. The pH and temperature optima for the synthetase are 6.0 and 37 degrees C, respectively. The apparent Km for the substrates ATP, biotin, and apo 1.3 S subunit of apotranscarboxylase are 38, 2.0, and 0.9 microM, respectively. Ni2+, Co2+, Zn2+, or Mn2+ could replace Mg2+ in the reaction. The affinity of synthetase toward metals is as follows: Zn2+ greater than Ni2+ greater than Mn2+ greater than Co2+ greater than Mg2+, and the activity with Zn2+ was much greater than that with the other divalent metals. EDTA completely inactivates the enzyme. The metals are necessary not only for the catalytic activity but also for the storage stability of the enzyme. The synthetase shows absolute specificity toward ATP.
通过在DE - 52纤维素和CM - 纤维素上进行离子交换色谱法，将将生物素连接到转羧酶脱辅基亚基上的合成酶（生物素-[甲基丙二酰辅酶A - 羧基转移酶]连接酶）（EC 6.3.4.9）纯化至同质。该合成酶是分子量为30,000的单体。该合成酶的最适pH和温度分别为6.0和37℃。脱辅基转羧酶的底物ATP、生物素和脱辅基1.3S亚基的表观Km分别为38、2.0和0.9μM。Ni2 +、Co2 +、Zn2 +或Mn2 +可在反应中替代Mg2 +。合成酶对金属的亲和力如下：Zn2 +＞Ni2 +＞Mn2 +＞Co2 +＞Mg2 +，并且Zn2 +存在时的活性远高于其他二价金属存在时的活性。EDTA可使该酶完全失活。金属不仅对催化活性是必需的，而且对酶的储存稳定性也是必需的。该合成酶对ATP表现出绝对特异性。
