Skarstedt M T, Silverstein E
J Biol Chem. 1976 Nov 10;251(21):6775-83.
The equilibrium constant of the reaction catalyzed by acetate kinase (E.C. 2.7.2.1.) was determined: K = (MgADP) (acetylphosphate)/(MgATP)(acetate) = 6.7 +/- 1.3 X 10 (-4) (pH 7.4, 25 degrees). The respective free nucleotides uncomplexed to magnesium inhibit the net reaction in both directions: competitively with the respective magnesium nucleotide (MgATP or MgADP) and noncompetitively with the co-substrate (acetate or acetylphosphate). Excess free magnesium also inhibits the net reaction in both directions. The inhibition is not competitive with the phosphoryl donor (MgATP or acetylphosphate) but is competitive with respect to the phosphoryl acceptor (acetate or MgADP). A 50-fold increase in concentration of reactants at equilibrium in 0.1 M Tris/HCl, pH 7.4, at 25 degrees resulted in a rise to plateau levels of the acetate equilibrium acetylphosphate exchange rate (measured with [U-14C]acetate) and of the ATP equilibrium ADP exchange rate (measured with [U-14C]ADP and 10-fold higher than acetate equilibrium acetylphosphate), suggesting that there is no compulsory order of binding of magnesium nucleotide and co-substrate and that all chemical transformation steps cannot be much slower than the dissociation steps. The ATP equilibrium ADP exchange rate was independent of the presence or concentration of co-substrate, whereas the acetate equilibrium acetylphosphate exchange reaction occurred only in the presence of magnesium nucleotide, and the rate was directly related to the degree of saturation of enzyme with magnesium nucleotide. The independent ATP equilibrium ADP exchange, which presumably involves a phosphoenzyme intermediate, was progressively inhibited by increasingly elevated MgADP concentrations (when MgADP/MgATP greater than or equal 5), but increasing MgATP/MgADP was not inhibitory, suggesting that MgADP may bind to nonphosphorylated as well as phosphorylated enzyme, but that MgATP cannot bind to phosphorylated enzyme. While direct transfer of the phosphoryl group between nucleotide and co-substrate in a concerted mechanism has not been ruled out, an "activated ping pong" mechanism can also be proposed which is compatible with the isotopic exchange and initial net rate kinetic results. This mechanism includes a phosphoenzyme intermediate and requires enzyme-bount MgADP for phosphorylation of the enzyme by acetylphosphate.
测定了由乙酸激酶（酶编号2.7.2.1.）催化的反应的平衡常数：K = （MgADP）（乙酰磷酸）/（MgATP）（乙酸） = 6.7 ± 1.3×10⁻⁴（pH 7.4，25℃）。未与镁络合的各自游离核苷酸在两个方向上均抑制净反应：与各自的镁核苷酸（MgATP或MgADP）竞争，与共底物（乙酸或乙酰磷酸）非竞争。过量的游离镁在两个方向上也抑制净反应。该抑制作用与磷酰基供体（MgATP或乙酰磷酸）无竞争性，但与磷酰基受体（乙酸或MgADP）有竞争性。在25℃下，0.1 M Tris/HCl，pH 7.4中平衡时反应物浓度增加50倍，导致乙酸平衡乙酰磷酸交换速率（用[U-¹⁴C]乙酸测定）和ATP平衡ADP交换速率（用[U-¹⁴C]ADP测定且比乙酸平衡乙酰磷酸高10倍）上升至平稳水平，这表明镁核苷酸和共底物的结合没有强制顺序，且所有化学转化步骤不会比解离步骤慢很多。ATP平衡ADP交换速率与共底物的存在或浓度无关，而乙酸平衡乙酰磷酸交换反应仅在镁核苷酸存在时发生，且速率与酶被镁核苷酸饱和的程度直接相关。推测涉及磷酰化酶中间体的独立ATP平衡ADP交换，随着MgADP浓度的不断升高（当MgADP/MgATP大于或等于5时）逐渐受到抑制，但增加MgATP/MgADP没有抑制作用，这表明MgADP可能与未磷酸化以及磷酸化的酶结合，但MgATP不能与磷酸化的酶结合。虽然尚未排除在协同机制中核苷酸和共底物之间磷酰基的直接转移，但也可以提出一种“活化乒乓”机制，该机制与同位素交换和初始净速率动力学结果相符。该机制包括一个磷酰化酶中间体，并且需要酶结合的MgADP用于乙酰磷酸对酶的磷酸化。
