Seminars Archive

Rik Wierenga

Abstract


Monday, January 29, 2000, 11:00
Seminar Room, ground floor, Building "T"
Sincrotrone Trieste, Basovizza
Structural enzymology of CoA dependent enzymes

Rik Wierenga
(Department of Biochemistry, University of Oulu, Linnanmaa,Oulu, Finland) ABSTRACT The contribution will focus on the structure-function relationship of enzymes of the hydratase/isomerase family and in particular on thiolases. Thiolases are a family of enzymes with high sequence similarity. They can be grouped into several classes such as the dimeric and tetrameric degradative thiolases and the tetrameric biosynthetic thiolases. These thiolases all catalyse the same reaction. The degradative thiolases catalyse the last step of the b-oxidation pathway which is a thiolytic cleavage reaction by which a 3-ketoacyl-CoA molecule is converted into acetyl-CoA and acyl-CoA; the biosynthetic thiolases, part of biosynthetic pathways, catalyse the reverse reaction, specifically the condensation of two acetyl-CoA molecules into acetoacetyl-CoA. The biosynthetic thiolases only work on small fatty acid tails (at most 4 carbon atoms), whereas the degradative thiolases can work on a wide range of fatty acid CoA molecules of which the carbon chain length can vary from 4 carbons to 20 carbons. Almost all enzymological data have been collected from tetrameric thiolases, both degradative and biosynthetic. Large differences in kinetic properties between the two classes of thiolases have been found, in particular with respect to the nature of the rate limiting steps as well as with respect to the reaction rates. It is well established that the reaction mechanism of all thiolases consist of two steps; first the enzyme is acylated at a cysteine in the active site. In the second step this acyl group is transferred to CoA (by the degradative thiolases) or acetylCoA (by the biosynthetic thiolases). Crystal structures are known of the dimeric degradative thiolase (unliganded) (JMB 273, 1997, 714-728) and of the tetrameric biosynthetic thiolase (liganded). Using flash freezing techniques two reaction intermediates of the biosynthetic thiolase have been characterised. It concerns the complex of CoA with the acetylated enzyme (Structure 7, 1999, 1279-1290) and of the complex of acetyl-CoA with the acetylated enzyme (submitted). These structures enlighten several important features of the reaction mechanisms of these enzymes, such as (i) the importance of a hydrogen bonding network which links the catalytic residues and which extends through the center of the molecule towards the backside of the molecule and (ii) the geometry of the active site allowing acetyl-CoA to be an electrophilic reactant (acetylation of a cysteine) as well as a nucleophilic reactant (in the condensation reaction).