SUBUNIT INTERFACE MUTANTS OF RABBIT MUSCLE ALDOLASE FORM ACTIVE DIMERS

We report the construction of subunit interface mutants of rabbit musc le aldolase A with altered quaternary structure. A mutation has been d escribed that causes nonspherocytic hemolytic anemia and produces a th ermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8 623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site -directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, G in, or Val. Rabbit aldolase D128G purified from Escherichia coli is fo und to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wildtype rabbit enzyme in secondary structure and kinetic propert ies. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species e xists for the mutants. In sedimentation velocity experiments, the muta nt enzymes exist as mixtures of dimer and tetramer at 4 degrees C. Sed imentation at 20 degrees C shows that the mutant enzymes are >99.5% di meric and, in the presence of substrate, that the dimeric species is a ctive. Differential scanning calorimetry demonstrates that T,values of the mutant enzymes are decreased by 12 degrees C compared to wild-typ e enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldo lase is to provide thermostability.