Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Aspartate residues function as proton accepters in catalysis and are involv ed in ionic interactions stabilizing subunit assembly. In an attempt to unr avel the role of a conserved aspartate (D89) in sheep-liver tetrameric seri ne hydroxymethyltransferase (SHMT), it was converted into aspargine by site -directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers an d tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5'-phosphate (PLP) concentration used during purification. T he D89N mutant tetramer was as active as the wild-type enzyme and had simil ar kinetic and spectral properties in the presence of 500 mu M PLP. The qui nonoid spectral intermediate commonly seen in the case of SHMT was also see n in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 mm, it had a negligible visible CD spectrum at 425 nm and was only 5 % active. The apo-D89N mutant tetramer was a dimer unlike the ape-form of t he wild-type enzyme which was present predominantly as a tetramer. Furtherm ore the ape mutant dimer could not be reconstituted to the hole-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cyt osolic recombinant SHMT indicates that this residue (D90 in the human enzym e) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry re lated subunit forming the tight dimer. D89 is at the interface of tight dim ers where the PLP 5'-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in dec reased affinity of the enzyme for the cofactor.