Aminolaevulinic acid synthase of Rhodobacter capsulatus: high-resolution kinetic investigation of the structural basis for substrate binding and catalysis.

The first enzyme of haem biosynthesis, ALAS (5-aminolaevulinic acid synthase), catalyses the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to 5-aminolaevulinic acid, CO(2) and CoA. The crystal structure of Rhodobacter capsulatus ALAS provides the first snapshots of the structural basis for substrate binding and catalysis. To elucidate the functional role of single amino acid residues in the active site for substrate discrimination, substrate positioning, catalysis and structural protein rearrangements, multiple ALAS variants were generated. The quinonoid intermediates I and II were visualized in single turnover experiments, indicating the presence of an α-amino-β-oxoadipate intermediate. Further evidence was obtained by the pH-dependent formation of quinonoid II from the product 5-aminolaevulinic acid. The function of Arg(21), Thr(83), Asn(85) and Ile(86), all involved in the co-ordination of the succinyl-CoA substrate carboxy group, were analysed kinetically. Arg(21), Thr(83)and Ile(86), all of which are located in the second subunit to the intersubunit active site, were found to be essential. Their location in the second subunit provides the basis for the required structural dynamics during the complex condensation of both substrates. Utilization of L-alanine by the ALAS variant T83S indicated the importance of this residue for the selectiveness of binding with the glycine substrate compared with related amino acids. Asn(85) was found to be solely important for succinyl-CoA substrate recognition and selectiveness of binding. The results of the present study provide a novel dynamic view on the structural basis of ALAS substrate-binding and catalysis.