No abstract

Five single replacement mutants of catalase A fromSaccharomyces cerevisiae were prepared (F148V, F149V, F156V, FI59V, and VIllA). The exchanges were expected to relieve steric constraints in the lowest part of the major substrate channel. The overall stability of the isolated enzymes is unaffected by the respective amino acid exchanges, but some modifications lead to decreased protohaem binding. AH isolated mutants (most pronounced the VillA-species) show decreased catalatic and markedly increased peroxidatic activity, both with aliphatic and aromatic substrates. These effects can in part be explained by steric effects, but also reveal destabilisation of compound I.

Abstract:Yeast peroxisomal catalase A, obtained at high yields by over expression of the C-terminally modified gene from a 2p-plasmid, has been crystallized in a form suitable for high resolution X-ray diffraction studies. Brownish crystals with bipyrimidal morphology and reaching ca. 0.8 mm in size were produced by the hanging drop method using ammonium sulphate as precipitant. These crystals diffract better than 2.0 8, resolution and belong to the hexagonal space group P6122 with unit cell parameters a = b = 184.3 8, and c = 305.5 A. An X-ray data set with 76% completeness at 3.2 A resolution was collected in a rotating anode generator using mirrors to improve the collimation of the beam. An initial solution was obtained by molecular replacement only when using a beef liver catalase tetramer model in which fragments with no sequence homology had been omitted, about 150 residues per subunit. In the structure found a single molecule of catalase A (a tetramer with accurate 222 molecular symmetry) is located in the asymmetric unit of the crystal with an estimated solvent content of about 61%. The preliminary analysis of the structure confirms the absence of a carboxy terminal domain as the one found in the catalase from Penicillium vitulue, the only other fungal catalase structure available. The NADPH binding site appears to be involved in crystal contacts, suggesting that heterogeneity in the occupancy of the nucleotide can be a major difficulty during crystallization.