Selective modification of the active center of renal iodothyronine 5'-deiodinase by iodoacetate.

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.