MODULATION OF THE ACTIVITY OF MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE H352C BY THE REDOX STATE OF THE ENGINEERED INTERDOMAIN DISULFIDE BOND

Molecular modeling suggested that the large and small domain of mitoch ondrial aspartate aminotransferase might be linked by an engineered di sulfide bond that could be expected to interfere with ligand-induced a nd syncatalytic changes in conformation and thus to assist in the eluc idation of their significance for the catalytic mechanism. His-352, wh ich is situated in the small domain close to Cys-166 of the large doma in, was replaced with a cysteine residue by oligonucleotide-directed m utagenesis. Aspartate aminotransferase H352C, that had not been expose d to reducing conditions, in part contained a disulfide bond between C ys-166 and Cys-352. Exposure to a reducing agent cleaved the cross-lin k completely and produced an enzyme derivative with 8% of the activity of the wild type enzyme. Cu2+-mediated autoxidation resulted in compl ete formation of the disulfide bond and a decrease in enzymic activity to 2%. Independently of the redox state of the disulfide bond, the H3 52C substitution seems to shift the equilibrium from the open toward t he closed conformation of the enzyme. This change in conformation was accompanied by an increase in the binding affinity for both the amino and oxo acid substrate by one order of magnitude. Apparently, 1-2 kcal /mol of the binding energy of the substrates are no longer diverted to shift the conformational equilibrium toward the closed conformation. The k(cat)/K-m values were unchanged or even increased in the reduced form of the mutant enzyme and only slightly decreased in its oxidized form, Both the disulfide-independent decrease in enzymic activity, as observed in reduced aspartate aminotransferase H352C and also in two o ther mutant enzymes (C166H/H352C and H352Q), and the redox-dependent m odulation of activity indicate that unhindered domain movements are es sential for full catalytic competence of aspartate aminotransferase.