Biomedical Engineering Reference
In-Depth Information
CHAPTER 1
METHODS OF INVESTIGATION OF THE STRUCTURE AND ACTION
MECHANISMS OF ACTIVE SITES OFENZYMES
1.1. Physical methods
1.1.1.
X-RAY STRUCTURAL ANALYSIS
The traditional approach to the investigation of intermediates of an enzymatic reaction
involves the X-ray structural analysis of enzyme complexes and substrate analogs.
Recently three different crystallographic techniques to trap enzyme bound intermediates
in a crystal lattice have been proposed. The first technique uses ultrafast kinetics
methods to obtain complete data sets on the enzymatic time scale (Bolduc et al., 1995;
Stoddard, 1996). One successful strategy is flash-photolysis inducing the homogeneous
synchronization cycle in an enzyme crystal with a photoactive substrate analog. As a
result the enzyme-substrate complex is formed. As an another strategy, site-directed
mutagenesis of key catalytic residues creates a kinetic bottleneck at specific steps which
can be used to determine the structure of distinct intermediates. These methods have
been used to determine the structure of intermediates in isocitrate dehydrogenase.
1
Search WWH ::
Custom Search