Ballicora M A, Wolosiuk R A
Instituto de Investigaciones Bioquímicas Fundación Campomar, IIBBA-CONICET, FCEN-UBA, Buenos Aires, Argentina.
Eur J Biochem. 1994 Jun 1;222(2):467-74. doi: 10.1111/j.1432-1033.1994.tb18887.x.
To characterize the mechanism of chloroplast fructose-1,6-bisphosphatase activation, we have examined kinetic and structural changes elicited by protein perturbants and reductants. At variance with its well-known capacity for enzyme inactivation, 150 mM sodium trichloroacetate yielded an activatable chloroplast fructose-1,6-bisphosphatase in the presence of 1.0 mM fructose 1,6-bisphosphate and 0.1 mM Ca2+. Other sugar bisphosphates did not replace fructose 1,6-bisphosphate whereas Mg2+ and Mn2+ were functional in place of Ca2+. Variations of the emission fluorescence of intrinsic fluorophores and a noncovalently bound extrinsic probe [2-(p-toluidinyl)naphthalene-6-sulfonate] indicated the presence of conformations different from the native form. A similar conclusion was drawn from the analysis of absorption spectra by means of fourth-derivative spectrophotometry. The effect of these conformational changes on the reductive process was studied by subsequently incubating the enzyme with dithiothreitol. The reaction of chloroplast fructose-1,6-bisphosphatase with dithiothreitol was accelerated 13-fold by the chaotropic anion: second-order rate constants were 48.1 M-1.min-1 and 3.7 M-1.min-1 in the presence and in the absence of trichloroacetate, respectively. Thus, the enhancement of the reductive activation by compounds devoid of redox activity illustrated that the modification of intramolecular noncovalent interactions of chloroplast fructose-1,6-bisphosphatase plays an essential role in the conversion of enzyme disulfide bonds to sulfhydryl groups. In consequence, a conformational change would operate concertedly with the reduction of disulfide bridges in the light-dependent activation mediated by the ferredoxin-thioredoxin system.
为了阐明叶绿体果糖-1,6-二磷酸酶激活的机制，我们研究了蛋白质干扰剂和还原剂引起的动力学和结构变化。与众所周知的酶失活能力不同，在1.0 mM果糖1,6-二磷酸和0.1 mM Ca2+存在的情况下，150 mM三氯乙酸产生了一种可激活的叶绿体果糖-1,6-二磷酸酶。其他糖二磷酸不能替代果糖1,6-二磷酸，而Mg2+和Mn2+可以替代Ca2+发挥作用。内在荧光团和非共价结合的外在探针[2-(对甲苯胺基)萘-6-磺酸盐]发射荧光的变化表明存在与天然形式不同的构象。通过四阶导数分光光度法对吸收光谱的分析也得出了类似的结论。随后用二硫苏糖醇孵育该酶，研究了这些构象变化对还原过程的影响。离液序列高的阴离子使叶绿体果糖-1,6-二磷酸酶与二硫苏糖醇的反应加速了13倍：在存在和不存在三氯乙酸的情况下，二级速率常数分别为48.1 M-1·min-1和3.7 M-1·min-1。因此，缺乏氧化还原活性的化合物增强还原激活作用表明，叶绿体果糖-1,6-二磷酸酶分子内非共价相互作用的改变在酶二硫键向巯基的转化中起重要作用。因此，在铁氧还蛋白-硫氧还蛋白系统介导的光依赖性激活中，构象变化将与二硫键的还原协同作用。
