Harold J. Evans, Ralph B. Clark, Sterling A. Russell
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It has been confirmed that the first step of the overall acetic thiokinase reaction, assayed by determining the rate of synthesis of ATP from adenyl acetate and Tris pyrophosphate, requires Mg<sup>2+</sup>. This reaction does not require univalent cations. In contrast, the second step of the overall reaction in which acetyl-CoA and AMP are synthesized from adenyl acetate and CoA, requires univalent cations but is inhibited by Mg<sup>2+</sup> at a concentration of 0.01 M.</p></span></li><li><span>3.</span><span><p>3. In most experiments it has not been possible to dialyze the yeast acetic thiokinase sufficiently to show an absolute univalent cation requirement for either the overall reaction or the step in which acetyl-CoA and AMP are synthesized from adenyl acetate and CoA. Dialyzed enzyme extracts generally contain from 6·10<sup>−3</sup> to 8·10<sup>−3</sup> M K<sup>+</sup> which is sufficient to account for the activity in the absence of added univalent cations.</p></span></li><li><span>4.</span><span><p>4. The addition of KCl to the dialyzed acetic thiokinase greatly influences the maximum velocity of the reaction in which acetyl-CoA and AMP are formed from adenyl acetate and CoA. The addition of KCl to reactions, however, fails to measurably affect the <em>K</em><sub>m</sub> values for either adenyl acetate or CoA. It is concluded that KCl influences either the rate of breakdown of an enzyme-adenyl acetate-CoA complex or the number of active enzyme sites involved in the synthesis of acetyl-CoA and AMP from adenyl acetate and CoA.</p></span></li></ul></div>","PeriodicalId":100170,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects","volume":"92 3","pages":"Pages 582-594"},"PeriodicalIF":0.0000,"publicationDate":"1964-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0926-6569(64)90018-5","citationCount":"9","resultStr":"{\"title\":\"Cation requirements for the acetic thiokinase from yeast\",\"authors\":\"Harold J. Evans, Ralph B. Clark, Sterling A. Russell\",\"doi\":\"10.1016/0926-6569(64)90018-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p></p><ul><li><span>1.</span><span><p>1. Acetic thiokinase (acetate: CoA ligase (AMP), EC 6.2.1.1) has been partially purified from yeast and the effects of Mg<sup>2+</sup> and univalent cations on the overall reaction catalyzed by the enzyme and the individual steps of the reaction have been investigated. In addition of Mg<sup>2+</sup> the dialyzed enzyme required K<sup>+</sup>, NH<sub>4</sub><sup>+</sup> or Rb<sup>+</sup> for the catalysis of the overall reaction. The concentrations of univalent cations required for optimum activity were in the range from 0.01 to 0.05 M. The addition of Cs<sup>+</sup> to reaction mixtures resulted in an intermediate stimulation of activity whereas Na<sup>+</sup> or Li<sup>+</sup> were relatively ineffective.</p></span></li><li><span>2.</span><span><p>2. It has been confirmed that the first step of the overall acetic thiokinase reaction, assayed by determining the rate of synthesis of ATP from adenyl acetate and Tris pyrophosphate, requires Mg<sup>2+</sup>. This reaction does not require univalent cations. In contrast, the second step of the overall reaction in which acetyl-CoA and AMP are synthesized from adenyl acetate and CoA, requires univalent cations but is inhibited by Mg<sup>2+</sup> at a concentration of 0.01 M.</p></span></li><li><span>3.</span><span><p>3. In most experiments it has not been possible to dialyze the yeast acetic thiokinase sufficiently to show an absolute univalent cation requirement for either the overall reaction or the step in which acetyl-CoA and AMP are synthesized from adenyl acetate and CoA. Dialyzed enzyme extracts generally contain from 6·10<sup>−3</sup> to 8·10<sup>−3</sup> M K<sup>+</sup> which is sufficient to account for the activity in the absence of added univalent cations.</p></span></li><li><span>4.</span><span><p>4. The addition of KCl to the dialyzed acetic thiokinase greatly influences the maximum velocity of the reaction in which acetyl-CoA and AMP are formed from adenyl acetate and CoA. The addition of KCl to reactions, however, fails to measurably affect the <em>K</em><sub>m</sub> values for either adenyl acetate or CoA. 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引用次数: 9
摘要
1.1. 从酵母中部分纯化了乙酸硫激酶(醋酸辅酶a连接酶，EC 6.2.1.1)，并研究了Mg2+和一价阳离子对酶催化的整体反应和反应各步骤的影响。除了Mg2+外，透析酶还需要K+、NH4+或Rb+来催化整个反应。最佳活性所需的一价阳离子浓度在0.01 ~ 0.05 m之间。在反应混合物中加入Cs+对活性有中等刺激作用，而Na+或Li+则相对无效。通过测定乙酸腺苷和焦磷酸三酯合成ATP的速率，已经证实了整个乙酸硫激酶反应的第一步需要Mg2+。这个反应不需要一价阳离子。相反，由乙酸腺苷酯和辅酶a合成乙酰辅酶a和AMP的整个反应的第二步需要一价阳离子，但被浓度为0.01 M.3.3的Mg2+抑制。在大多数实验中，不可能充分地透析酵母醋酸硫激酶，以显示整个反应或从乙酸腺苷和辅酶a合成乙酰辅酶a和AMP的步骤需要绝对的单价阳离子。透析酶提取物通常含有6·10−3至8·10−3 M K+，这足以说明在没有添加一价阳离子的情况下的活性。醋酸腺苷酸和辅酶a生成乙酰辅酶a和AMP的最大反应速度受KCl的加入影响较大。然而，在反应中加入KCl对乙酸腺苷酯或辅酶a的Km值没有明显的影响。结果表明，KCl影响酶-乙酸腺苷-辅酶a复合物的分解速率或参与乙酸腺苷和辅酶a合成乙酰辅酶a和AMP的活性酶位点的数量。
Cation requirements for the acetic thiokinase from yeast
1.
1. Acetic thiokinase (acetate: CoA ligase (AMP), EC 6.2.1.1) has been partially purified from yeast and the effects of Mg2+ and univalent cations on the overall reaction catalyzed by the enzyme and the individual steps of the reaction have been investigated. In addition of Mg2+ the dialyzed enzyme required K+, NH4+ or Rb+ for the catalysis of the overall reaction. The concentrations of univalent cations required for optimum activity were in the range from 0.01 to 0.05 M. The addition of Cs+ to reaction mixtures resulted in an intermediate stimulation of activity whereas Na+ or Li+ were relatively ineffective.
2.
2. It has been confirmed that the first step of the overall acetic thiokinase reaction, assayed by determining the rate of synthesis of ATP from adenyl acetate and Tris pyrophosphate, requires Mg2+. This reaction does not require univalent cations. In contrast, the second step of the overall reaction in which acetyl-CoA and AMP are synthesized from adenyl acetate and CoA, requires univalent cations but is inhibited by Mg2+ at a concentration of 0.01 M.
3.
3. In most experiments it has not been possible to dialyze the yeast acetic thiokinase sufficiently to show an absolute univalent cation requirement for either the overall reaction or the step in which acetyl-CoA and AMP are synthesized from adenyl acetate and CoA. Dialyzed enzyme extracts generally contain from 6·10−3 to 8·10−3 M K+ which is sufficient to account for the activity in the absence of added univalent cations.
4.
4. The addition of KCl to the dialyzed acetic thiokinase greatly influences the maximum velocity of the reaction in which acetyl-CoA and AMP are formed from adenyl acetate and CoA. The addition of KCl to reactions, however, fails to measurably affect the Km values for either adenyl acetate or CoA. It is concluded that KCl influences either the rate of breakdown of an enzyme-adenyl acetate-CoA complex or the number of active enzyme sites involved in the synthesis of acetyl-CoA and AMP from adenyl acetate and CoA.
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