THE 1.8 ANGSTROM CRYSTAL-STRUCTURE OF THE DIMERIC PEROXISOMAL 3-KETOACYL-COA THIOLASE OF SACCHAROMYCES-CEREVISIAE - IMPLICATIONS FOR SUBSTRATE-BINDING AND REACTION-MECHANISM

The dimeric, peroxisomal 3-ketoacyl-CoA thiolase catalyses the convers ion of 3-ketoacyl-CoA into acyl-CoA, which is shorter by two carbon at oms. This reaction is the last step of the beta-oxidation pathway. The crystal structure of unliganded peroxisomal thiolase of the yeast Sac charomyces cerevisiae has been refined at 1.8 Angstrom resolution. An unusual feature of this structure is the presence of two helices, comp letely buried in the dimer and sandwiched between two beta-sheets. The analysis of the structure shows that the sequences of these helices a re not hydrophobic, but generate two amphipathic helices. The helix in the N-terminal domain exposes the polar side-chains to a cavity al th e dimer interface, filled with structured water molecules. The central helix in the C-terminal domain exposes its polar residues to an inter ior polar pocket. The refined structure has also been used to predict the mode of binding of the substrate molecule acetoacetyl-CoA, as well as the reaction mechanism. From previous studies it is known that Cys 125, His375 and Cys403 are important catalytic residues. In the propos ed model the acetoacetyl group fits near the two catalytic cysteine re sidues, such that the oxygen atoms point towards the protein interior. The distance between SG(Cys125) and C3(acetoacetyl-CoA) is 3.7 Angstr om. The O2 atom of the docked acetoacetyl group makes a hydrogen bond to N(Gly405), which would favour the formation of the covalent bond be tween SG(Cys125) and CS(acetoacetyl-CoA) of the intermediate complex o f the two-step reaction. The CoA moiety is proposed to bind in a groov e on the surface of the protein molecule. Most of the interactions of the CoA molecule are with atoms of the loop domain. The three phosphat e groups of the CoA moiety are predicted. to interact with side-chains of lysine and arginine residues, which are conserved in the dimeric t hiolases. (C) 1997 Academic Press Limited.