Garza-Ramos G, Pérez-Montfort R, Rojo-Domínguez A, de Gómez-Puyou M T, Gómez-Puyou A
Departamento de Bioenergética, Universidad Nacional Autónoma de México, México.
Eur J Biochem. 1996 Oct 1;241(1):114-20. doi: 10.1111/j.1432-1033.1996.0114t.x.
The possibility of using non-conserved amino acid residues to produce selective inhibition of homologous enzymes from different species has been further explored with triosephosphate isomerase. S-phenyl-p-toluenethiosulfonate (MePhSO2-SPh), which produces phenyl disulfides with accessible Cys residues, inhibits the activity of rabbit triosephosphate isomerase. The inhibition is due to derivatization of one of the five Cys residues of rabbit triosephosphate isomerase. The effect of MePhSO2-SPh on triosephosphate isomerase from Saccharomyces cerevisiae, Escherichia coli, chicken and Schizosaccharomyces pombe was also determined. MePhSO2-SPh did not affect the activity of triosephosphate isomerase from S. cerevisiae and E. coli but it inhibited triosephosphate isomerase from chicken and S. pombe. From an analysis of the Cys content of the various triosephosphate isomerases, it was evident that amongst the ones studied only those that have a Cys in position 217 (or in an equivalent position) were sensitive to MePhSO2-SPh. Methyl metanethiosulfonate (MeSO2-SMe), which produces methyl disulfides, had no effect on triosephosphate isomerases that lack Cys217 (S. cerevisiae and E. coli). In triosephosphate isomerases that have Cys217, MeSO2-SMe inhibited by 40-50% the activity of that from S. pombe, 20-25% that from rabbit but had no effect on the chicken enzyme. In the three latter triosephosphate isomerases, MeSO2-SMe protected against the strong inhibiting action of MePhSO2-SPh. The latter observations suggest that MeSO2-SMe and MePhSO2-SPh derivatize the same Cys and that significant inhibition of activity requires perturbation by the relatively large phenyl group. The intrinsic fluorescence of rabbit triosephosphate isomerase that had been derivatized to a phenyl disulfide was almost identical to that of the native enzyme. Thus, modification of Cys217 did not produce gross structural alterations, albeit it brought about important kinetic alterations, i.e. a nearly fivefold increase in the K(m) for glyceraldehyde 3-phosphate and a 65% decrease in Vmax. The effect of derivatizating Cys217 differs markedly from that produced by derivatization of Cys14 (another non-conserved cysteine). The differences may be explained from their position in the three-dimensional structure of the enzyme.
利用非保守氨基酸残基对来自不同物种的同源酶产生选择性抑制的可能性，已通过磷酸丙糖异构酶得到进一步探索。S-苯基-对甲苯硫代磺酸盐（MePhSO2-SPh）能与可及的半胱氨酸残基生成苯基二硫化物，它可抑制兔磷酸丙糖异构酶的活性。这种抑制是由于兔磷酸丙糖异构酶的五个半胱氨酸残基之一发生了衍生化。还测定了MePhSO2-SPh对酿酒酵母、大肠杆菌、鸡和粟酒裂殖酵母的磷酸丙糖异构酶的影响。MePhSO2-SPh不影响酿酒酵母和大肠杆菌的磷酸丙糖异构酶的活性，但能抑制鸡和粟酒裂殖酵母的磷酸丙糖异构酶。通过对各种磷酸丙糖异构酶的半胱氨酸含量分析可知，在所研究的酶中，只有那些在217位（或等效位置）含有半胱氨酸的酶对MePhSO2-SPh敏感。生成甲基二硫化物的甲硫代磺酸甲酯（MeSO2-SMe），对缺乏Cys217的磷酸丙糖异构酶（酿酒酵母和大肠杆菌）没有影响。在含有Cys217的磷酸丙糖异构酶中，MeSO2-SMe可使粟酒裂殖酵母的该酶活性抑制40 - 50%，兔的该酶活性抑制20 - 25%，但对鸡的该酶没有影响。在后面这三种磷酸丙糖异构酶中，MeSO2-SMe可防止MePhSO2-SPh的强烈抑制作用。后面这些观察结果表明，MeSO2-SMe和MePhSO2-SPh衍生化的是同一个半胱氨酸，而且活性的显著抑制需要相对较大的苯基产生扰动。衍生化为苯基二硫化物的兔磷酸丙糖异构酶的固有荧光与天然酶几乎相同。因此，Cys217的修饰虽未产生总体结构改变，尽管它带来了重要的动力学改变，即磷酸甘油醛的K(m)增加近五倍，Vmax降低65%。Cys217衍生化的效果与Cys14（另一个非保守半胱氨酸）衍生化产生的效果明显不同。这些差异可以从它们在酶的三维结构中的位置来解释。
