Kinetic consequences of engineering an "untethered" redox center in mycobacterium lepr'ae thioredoxin reductase/thioredoxin

llyeobactrium leprae contains a unique thioredoxin reductase (TR), in whi ts cognate electron accepter, thioredoxin (Tx), is covalent ly atached via mino acid linker. This hybrid enzyme (TR/Tx) has been cloned fiom g nomic .U. lepz DNA, expressed in E. eoli, and t)uriiied to homogeneity. T Enzynle catalyzes the NADPil-dependent reduction of boh I)TNB and 1, aaphthoquinone (NQ). Treatment of the enzyme with iodoacetamide results ;he abolition of DTNB reducing activity, but has no effect on NQ reductio mggesting that this activity does not require thioredoxin. To confirm our h )othesis we have engineered constructs for expressing separately Tit and T l'he 'untethered' enzyme (TR) catalyzes he reduction of 1)TNB ten tim dower than the hybrid enzyme , but the reduclion of NQ is equivalent to th :atalyzed by hybrid enzyme. The ptI dependences of DTNB reduction are d hrent for the hybrid and 'untethered' enzyme h)rms. The kineti(: and isotop :onsequences of 'untethering' these two redox components will be compar tad discussed, and a chernica.l mechanism will be presented as a framework fl he interi)retation of these i'eslllts. This work was supported by (;M334:19.