Reaction Mechanism of Thioredoxin: 3′‐Phospho‐adenylylsulfate Reductase Investigated by Site‐Directed Mutagenesis

Abstract

Properties of purified recombinant adenosine 3′‐phosphate 5′‐phosphosulfate (PAdoPS) reductase from Escherichia coli were investigated. The Michaelis constants for reduced thioredoxin and PAdoPS are 23 μM and 10 μM, respectively; the enzyme has a Vmax of 94–99 (μmol min−1 mg−1 and a molecular activity/catalytically active dimer of 95 s−1. Adenosine 3′,5′‐bisphosphate (PAdoP) inhibits competetively (Ki4 μM) with respect to PAdoPS; 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 PAdoPS 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 (ΔcysHIJ). Homogenous Cys239Ser and Tyr209Phe mutant PAdoPS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced Vmax by a factor of 4.5×103 (Vmax= 0.02–0.013 μmol min−1 mg−1) with marginal effects on Km for PAdoPS (19 μM) and reduced thioredoxin (14 μM). Mutation of Tyr209 drastically affected saturation with thioredoxin (Km 1.5 μM) and decreased Vmax (0.22–0.25 μmol min−1 mg−1) in addition to a small increase in Km for PAdoPS (31 μM). Chromophores as prosthetic groups were absent from recombinant PAdoPS 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 (Δλmax 292 nm) appears to be involved in the reduction. The data suggest a special ping‐pong mechanism with PAdoPS reacting with the reduced enzyme isomer in a Theorell‐Chance type mechanism. Copyright © 1995, Wiley Blackwell. All rights reserved