Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site-specific mutants

The Pyrococcus furiosus ornithine carbamoyltransferase (OTCase) is extremel y heat stable and maintains 50% of its catalytic activity after 60 min at 1 00 degreesC. The enzyme has an unusual quaternary structure when compared t o anabolic OTCases from mesophilic organisms. It is built up of four trimer s arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main in terfaces that occur between the catalytic trimers within the dodecamer. The y participate in either hydrophobic clusters or ionic interactions. In orde r to elucidate the role played by the oligomerization in the enzyme stabili ty at very high temperatures, we performed mutagenesis studies of these res idues. All the variants show similar catalytic activities and kinetic prope rties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. Th e variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering mor e drastically the interfaces results in a proportional decrease in heat sta bility, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat sta bility and is a trimer. These results suggest that extreme thermal stabiliz ation of this OTCase is achieved in part through oligomerization.