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

Iovila Marcinkeviciene, John S. Blanchard

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997
Externally publishedYes

Fingerprint

Thioredoxin-Disulfide Reductase
Thioredoxins
Mycobacterium
Oxidation-Reduction
engineering
kinetics
Kinetics
Enzymes
enzymes
Dithionitrobenzoic Acid
enzymatic treatment
Iodoacetamide
electrons
Electrons
acids
DNA
Acids

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Kinetic consequences of engineering an "untethered" redox center in mycobacterium lepr'ae thioredoxin reductase/thioredoxin. / Marcinkeviciene, Iovila; Blanchard, John S.

In: FASEB Journal, Vol. 11, No. 9, 1997.

Research output: Contribution to journalArticle

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