REACTION-MECHANISM OF THIOREDOXIN - 3'-PHOSPHO-ADENYLYLSULFATE REDUCTASE INVESTIGATED BY SITE-DIRECTED MUTAGENESIS

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 mu M and 10 mu M, respectively; the enzyme has a V-max of 94-99 mu mol min(-1) mg(-1) and a molecular activity/catalytically active dimer of 95 s(-1). Adenosine 3',5'-bisphosphate (PAdoP) inhibits competitively (K-i 4 mu M) 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 (Delta cysHIJ). Homogenous Cys239Ser and Tyr209Phe mutant PAdoPS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced V-max by a factor of 4.5x 10(3) (V-max,, = 0.02-0.013 mu mol min(-1) mg(-1)) with marginal effects on K-m for PAdoPS (19 mu M) and reduced thioredoxin (14 mu M) Mutation of Tyr209 drastically affected saturation with thioredoxin (K-m 1.5 mu M) and decreased V-max (0.22-0.25 mu mol min(-1) mg(-1)) in addition to a small increase in K-m for PAdoPS (31 mu M). 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 (Delta lambda(max) 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.