Zubillaga R A, Pérez-Montfort R, Gómez-Puyou A
Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, México, D.F.
Arch Biochem Biophys. 1994 Sep;313(2):328-36. doi: 10.1006/abbi.1994.1395.
Triosephosphate isomerase from rabbit has 5 Cys and 2 Met, while triosephosphate isomerase from yeast has 2 Cys (present in the rabbit enzyme in equivalent positions) and no Met. Since chloramine-T oxidizes Cys and Met, we determined the effect it has on the activity and structure of both enzymes. The activity of triosephosphate isomerase from rabbit was more sensitive to chloramine-T than that of the yeast enzyme (under conditions where the rabbit isomerase was completely inactive, the yeast enzyme exhibited approximately 50% activity). An initial effect of chloramine-T on triosephosphate isomerase was the oxidation of Cys and the formation of catalytically active acidic isoforms. For the yeast isomerase, the two processes were slower. Our data suggest that oxidation of Cys 126, which is conserved in all of the studied species, does not abolish catalysis. Chloramine-T also oxidized the two Met of the rabbit enzyme. At ratios of 50 chloramine-T/monomer, circular dichroism studies showed that the rabbit enzyme, but not that from yeast, underwent extensive alterations of tertiary and secondary structures. This was accompanied by formation of stable dimers, whose cross-linking was not through disulfide bonds. Studies of dimer formation at various enzyme concentrations showed that cross-linking was between monomers of the same dimer. Under conditions that led to cross-linking, rabbit triosephosphate isomerase took up 2.7 mol of 3H from NaB3H4/mol dimer, and the yeast enzyme incorporated only 0.4 mol of 3H. Thus cross-linking was most likely via a Schiff base. The results revealed the points whose modification caused inactivation of the rabbit enzyme.
兔磷酸丙糖异构酶有5个半胱氨酸（Cys）和2个甲硫氨酸（Met），而酵母磷酸丙糖异构酶有2个半胱氨酸（在兔酶中处于相同位置）且没有甲硫氨酸。由于氯胺-T会氧化半胱氨酸和甲硫氨酸，我们测定了其对两种酶的活性和结构的影响。兔磷酸丙糖异构酶的活性比酵母酶对氯胺-T更敏感（在兔异构酶完全失活的条件下，酵母酶仍表现出约50%的活性）。氯胺-T对磷酸丙糖异构酶的初始作用是氧化半胱氨酸并形成具有催化活性的酸性同工型。对于酵母异构酶，这两个过程较慢。我们的数据表明，在所有研究物种中都保守的半胱氨酸126的氧化不会消除催化作用。氯胺-T还氧化了兔酶的两个甲硫氨酸。在氯胺-T/单体比例为50时，圆二色性研究表明，兔酶而非酵母酶的三级和二级结构发生了广泛改变。这伴随着稳定二聚体的形成，其交联不是通过二硫键。在不同酶浓度下对二聚体形成的研究表明，交联发生在同一二聚体的单体之间。在导致交联的条件下，兔磷酸丙糖异构酶每摩尔二聚体从硼氢化钠（NaB3H4）中摄取2.7摩尔的3H，而酵母酶仅掺入0.4摩尔的3H。因此，交联最有可能是通过席夫碱进行的。结果揭示了兔酶修饰导致失活的位点。
