Yoshida M, Allison W S
J Biol Chem. 1983 Dec 10;258(23):14407-12.
The soluble F1-ATPase from the thermophilic bacterium PS3 (TF1) contains no endogenous adenine nucleotides and contains about 0.2 g ions of Mg2+/mol which resists removal by repeated centrifugation-elution on columns of Sephadex G-50. The isolated enzyme will not bind additional Mg2+ added in the absence of adenine nucleotides nor is the rate of inactivation of the isolated enzyme by dicyclohexylcarbodiimide (DCCD) affected by the addition of Mg2+. When ADP is added to isolated TF1, a 1:1 TF1 X ADP complex is formed which is stable to repeated gel permeation on columns of Sephadex G-50 subjected to centrifugation-elution. On formation of the 1:1 TF1 X ADP complex, the rate of inactivation of the enzyme by DCCD is accelerated 6-fold. The rate of inactivation of the 1:1 TF1 X ADP complex by DCCD is not further stimulated in the presence of 2 mM ADP which indicates that the binding of ADP to a single site in the enzyme is sufficient to promote maximal stimulation of the inactivation. Addition of Mg2+ to the 1:1 TF1 X ADP complex results in the binding of about 1 g ion of Mg2+/mol of enzyme. The 1:1:1 TF1 X ADP X Mg2+ complex thus formed is sluggishly inactivated by DCCD. When the Mg2+ is removed from the TF1 X ADP X Mg2+ complex by treatment with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, the rate of inactivation of the enzyme by DCCD is accelerated 4-fold. Other divalent metal ions protect the 1:1 TF1 X ADP complex against inactivation by DCCD. Of these, Mn2+, Zn2+, Co2+, and Cd2+, which are about as equally effective as Mg2+ as cofactors for the hydrolytic reaction when present at 0.2 mM, offer about equal protection of the complex against inactivation by DCCD also when present at 0.2 mM. These results indicate that the binding site for ADP in the 1:1 TF1 X ADP complex is a catalytic site. TF1, inactivated by 92% with DCCD, has the same capacity to bind ADP as the active enzyme, forming a tight 1:1 TF1 X ADP complex which is stable to repeated centrifugation-elution on columns of Sephadex G-50. The 1:1 TF1 X ADP complex retains its capacity to bind Mg2+ to form the 1:1:1 TF1 X ADP X Mg2+ complex after it is inactivated by 88% with DCCD.
嗜热细菌PS3的可溶性F1 - ATP酶（TF1）不含内源性腺嘌呤核苷酸，每摩尔含有约0.2克离子的Mg2 +，通过在Sephadex G - 50柱上反复离心洗脱也难以去除。在没有腺嘌呤核苷酸的情况下，分离出的酶不会结合额外添加的Mg2 +，并且二环己基碳二亚胺（DCCD）使分离出的酶失活的速率也不受添加Mg2 +的影响。当向分离出的TF1中加入ADP时，会形成1:1的TF1×ADP复合物，该复合物在Sephadex G - 50柱上进行离心洗脱的反复凝胶渗透过程中是稳定的。在形成1:1的TF1×ADP复合物时，DCCD使酶失活的速率加快了6倍。在2 mM ADP存在的情况下，DCCD使1:1的TF1×ADP复合物失活的速率不再进一步受刺激，这表明ADP与酶中单个位点的结合足以促进失活的最大刺激。向1:1的TF1×ADP复合物中添加Mg2 +会导致每摩尔酶结合约1克离子的Mg2 +。由此形成的1:1:1的TF1×ADP×Mg2 +复合物被DCCD缓慢失活。当用反式 - 1,2 - 二氨基环己烷 - N,N,N',N' - 四乙酸处理从TF1×ADP×Mg2 +复合物中去除Mg2 +后，DCCD使酶失活的速率加快了4倍。其他二价金属离子可保护1:1的TF1×ADP复合物不被DCCD失活。其中，Mn2 +、Zn2 +、Co2 +和Cd2 +在浓度为0.2 mM时作为水解反应的辅因子与Mg2 +的效果大致相同，当它们也以0.2 mM存在时，对复合物不被DCCD失活的保护作用也大致相同。这些结果表明，1:1的TF1×ADP复合物中ADP的结合位点是一个催化位点。被DCCD失活92%的TF1与活性酶结合ADP的能力相同，形成紧密的1:1的TF1×ADP复合物，该复合物在Sephadex G - 50柱上进行反复离心洗脱时是稳定的。在被DCCD失活88%后，1:1的TF1×ADP复合物仍保留结合Mg2 +形成1:1:1的TF1×ADP×Mg2 +复合物的能力。
