Leonard J L, Visser T J
Biochim Biophys Acta. 1984 Jun 14;787(2):122-30. doi: 10.1016/0167-4838(84)90070-0.
Pretreatment of renal iodothyronine 5'-deiodinase with sulfhydryl reagents, iodoacetate, iodoacetamide and N-alkylmaleimides, results in irreversible loss of catalytic activity. Iodoacetate and iodoacetamide were the most potent inhibitors, being 100- to 1000-times more potent than N-alkylmaleimides. Iodoacetate and iodoacetamide inactivation followed pseudo-first-order kinetics with maximum inactivation rate constants of 1.56 min-1 and 0.87 min-1, respectively. Thyroxine and 3,3',5'-triiodothyronine and the competitive inhibitor iopanoate, protected the enzyme against iodoacetate inhibition. Protection by 3,3',5'-triiodothyronine was competitive with iodoacetate with a dissociation constant (Kd) of 113 nM; in close agreement with the Km for rT3 of 190 nM determined under similar reaction conditions. [3H]Carboxymethylation of renal membranes in the absence and presence of 3,3',5'-triiodothyronine showed specific incorporation of iodo[3H]acetate into substrate-protected sites of 35-40% of total when non-essential residues were first blocked with excess unlabeled iodoacetate. ' Protectable ' [3H]acetate incorporation followed pseudo-first-order kinetics and the rate constant for incorporation was identical to the rate constant for inactivation. These results indicate that iodoacetate fulfills the minimum criteria for an active-site-directed reagent for renal 5'-deiodinase and that a sulfhydryl group is in close proximity to the iodothyronine-binding site.
用巯基试剂、碘乙酸、碘乙酰胺和N - 烷基马来酰亚胺对肾碘甲状腺原氨酸5'-脱碘酶进行预处理，会导致催化活性不可逆丧失。碘乙酸和碘乙酰胺是最有效的抑制剂，其效力比N - 烷基马来酰亚胺高100至1000倍。碘乙酸和碘乙酰胺的失活遵循假一级动力学，最大失活速率常数分别为1.56 min⁻¹和0.87 min⁻¹。甲状腺素、3,3',5'-三碘甲状腺原氨酸和竞争性抑制剂碘番酸可保护该酶免受碘乙酸抑制。3,3',5'-三碘甲状腺原氨酸的保护作用与碘乙酸具有竞争性，解离常数（Kd）为113 nM；这与在类似反应条件下测定的反式三碘甲状腺原氨酸（rT3）的Km值190 nM非常一致。在不存在和存在3,3',5'-三碘甲状腺原氨酸的情况下，对肾膜进行[³H]羧甲基化显示，当用过量未标记的碘乙酸首先阻断非必需残基时，碘[³H]乙酸特异性掺入到占总量35 - 40%的底物保护位点。“可保护的”[³H]乙酸掺入遵循假一级动力学，掺入速率常数与失活速率常数相同。这些结果表明，碘乙酸符合作为肾5'-脱碘酶活性位点导向试剂的最低标准，并且一个巯基与碘甲状腺原氨酸结合位点紧密相邻。
