Meixner-Monori B, Kubicek C P, Röhr M
Can J Microbiol. 1984 Jan;30(1):16-22. doi: 10.1139/m84-003.
Pyruvate kinase from the filamentous, citric acid producing fungus Aspergillus niger was purified about 100-fold by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration. The addition of fructose-1,6-diphosphate was necessary to prevent loss of activity during purification. The enzyme purified in the presence of fructose-1,6-diphosphate (FDP) exhibits hyperbolic kinetics with respect to phosphoenolpyruvate (PEP) and ADP. Monovalent cations activated the enzyme (K+, NH4+). FDP neither activated nor inhibited the enzymatic activity from extracts freshly prepared in the absence of exogenous FDP; ATP showed a weak activation. In contrast the enzyme from crude extracts which had been stored in the presence of glycerol for 3 days showed activation by FDP or a metabolite thereof and inhibition by ATP. In the absence of FDP sigmoidal kinetics were obtained with respect to PEP, which became hyperbolic kinetics after addition of FDP. ATP inhibition turned into slight ATP activation in the presence of FDP. However, it was possible to reactivate inactivated pyruvate kinase (after dialysis in the absence of FDP) by adding FDP to the enzyme assay. From these results and because of the very high affinity of pyruvate kinase for FDP (Ka less than 0.1 microM), it is concluded that the enzyme probably has FDP bound to the protein in vivo. The significance of this hypothesis to the regulation of glycolysis in A. niger, with special reference to the mechanism of citric acid accumulation, is discussed.
通过硫酸铵沉淀、DEAE-纤维素色谱法和凝胶过滤，从丝状产柠檬酸真菌黑曲霉中纯化出丙酮酸激酶，纯化倍数约为100倍。在纯化过程中，添加1,6-二磷酸果糖对于防止活性丧失是必要的。在1,6-二磷酸果糖（FDP）存在下纯化的该酶，对磷酸烯醇丙酮酸（PEP）和ADP呈现双曲线动力学。单价阳离子激活该酶（K⁺、NH₄⁺）。FDP对在无外源FDP情况下新鲜制备的提取物中的酶活性既不激活也不抑制；ATP表现出微弱的激活作用。相反，在甘油存在下储存3天的粗提取物中的酶，表现出被FDP或其代谢产物激活以及被ATP抑制的特性。在没有FDP的情况下对PEP呈现S形动力学，添加FDP后变为双曲线动力学。在FDP存在下，ATP抑制转变为轻微的ATP激活。然而，通过在酶测定中添加FDP，可以使失活的丙酮酸激酶重新激活（在无FDP的情况下透析后）。根据这些结果，并且由于丙酮酸激酶对FDP具有非常高的亲和力（Ka小于0.1微摩尔），可以得出结论，该酶在体内可能有FDP与蛋白质结合。讨论了这一假设对黑曲霉糖酵解调节的意义，特别提及柠檬酸积累的机制。
