Open AccessReaction Mechanism of Thioredoxin: 3′‐Phospho‐adenylylsulfate Reductase Investigated by Site‐Directed Mutagenesis
Author(s) -
Berendt Uwe,
Haverkamp Thomas,
Prior Antje,
Schwenn Jens D.
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.347_1.x
Subject(s) - biochemistry , thioredoxin , site directed mutagenesis , phenylglyoxal , chemistry , active site , mutagenesis , enzyme , adenosine , escherichia coli , microbiology and biotechnology , thioredoxin reductase , stereochemistry , mutant , biology , amino acid , gene , arginine
Properties of purified recombinant adenosine 3′‐phosphate 5′‐phosphosulfate ( P Ado PS ) reductase from Escherichia coli were investigated. The Michaelis constants for reduced thioredoxin and P Ado PS are 23 μM and 10 μM, respectively; the enzyme has a V max of 94–99 (μmol min −1 mg −1 and a molecular activity/catalytically active dimer of 95 s −1 . Adenosine 3′,5′‐bisphosphate ( P Ado P ) inhibits competetively (K i 4 μM) with respect to P Ado PS ; adenosine 2′,5′‐bisphosphate and sulfite are not inhibitory. Alkylation by SH‐group inhibitors irreversibly inactivates the enzyme. The structural gene ( cysH ) encodes for a small polypeptide with a single Cys residue located in a conserved cluster (KXECGI/LH) of amino acids. Involvement of the only Cys and of Tyr209 in the reduction of P Ado PS to sulfite was investigated by site‐specific mutagenesis: cysH was mutated by single‐strand‐overlay extension PCR; the mutated genes were cloned in pBTac1 and expressed in E. coli RL 22 (Δ cysHIJ ). Homogenous Cys239Ser and Tyr209Phe mutant P Ado PS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced V max by a factor of 4.5×10 3 ( V max = 0.02–0.013 μmol min −1 mg −1 ) with marginal effects on K m for P Ado PS (19 μM) and reduced thioredoxin (14 μM). Mutation of Tyr209 drastically affected saturation with thioredoxin ( K m 1.5 μM) and decreased V max (0.22–0.25 μmol min −1 mg −1 ) in addition to a small increase in K m for P Ado PS (31 μM). Chromophores as prosthetic groups were absent from recombinant P Ado PS reductase. Difference absorption spectra between reduced and oxidized forms of wild‐type and mutated proteins indicated that, in addition to Cys239 and Tyr209, an unidentified Trp (Δλ max 292 nm) appears to be involved in the reduction. The data suggest a special ping‐pong mechanism with P Ado PS reacting with the reduced enzyme isomer in a Theorell‐Chance type mechanism.