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).