MULTINUCLEAR NMR RESONANCE ASSIGNMENTS AND THE SECONDARY STRUCTURE OFESCHERICHIA-COLI THIOESTERASE PROTEASE I - A MEMBER OF A NEW SUBCLASSOF LIPOLYTIC ENZYMES/

Escherichia coli thioesterase/protease I is a 183 amino acid protein w ith a molecular mass of 20 500. This protein belongs to a new subclass of lipolytic enzymes of the serine protease superfamily, but with a n ew GDSLS consensus motif, of which no structure has yet been determine d. The protein forms a tetramer at pH values above 6.5 and exists as a monomer at lower pH values. Both monomer and tetramer are catalytical ly active. From analysis of a set of heteronuclear multidimensional NM R spectra with uniform and specific amino acid labeled protein samples , we have obtained near-complete resonance assignments of the backbone H-1, C-13 and N-15 nuclei (BMRB databank accession number 4060), The secondary structure of E. coli thioesterase/protease I was further ded uced from the consensus chemical shift indices, backbone short-and med ium-range NOEs, and amide proton exchange rates. The protein was found to consist of four beta-strands and seven alpha-helices, arranged in alternate order. The four beta-strands were shown to form a parallel b eta-sheet. The topological arrangement of the beta-strands of -1x, +2x , +1x appears to resemble that of the core region of the alpha beta hy drolase superfamily, typically found in common lipases and esterases. However, substantial differences, such as the number of beta-strands a nd the location of the catalytic triad residues, make it difficult to give a definitive classification of the structure of E. coli thioester ase/protease I at present.