Structure and Activity (Reset) Press reset (veiwing the enzyme will be easier if you scroll the picture all the way to the right)
**Note-The model can be moved by the veiwer; however, these movements may cause problems in veiwing the individual pages and structures set up by the push buttons. It is suggested that the commands are used in order the first time through.
The quaternary structure of DLDH is made up of 2 identical chains, A and B. These two chains are held together by a network of noncovalent interactions between their backbones as well as side chains of their respected amino acids. It is interesting to note that the individual monomers are not symmetrical but that the dimer is symmetrical, as is the case with most proteins.
These types of interactions also help dictate the globular structures of enzymes as well as their secondary structures. The DLDH contains 6 total regions (three on each monomer) that are formed by beta sheets, four of which appear to be beta barrels that are each formed by an anti-parallel beta sheet and a beta-alpha-beta motif. The alpha-helices appear to be used as stabilizing links to uphold this hybrid beta-barrel construction; however, upon protein sequencing both the beta sheets and alpha-helices contain regions that interact with the FAD cofactor. (Reset)The other 2 beta sheet regions (one in each monomer) lie on the midline of the dimer and are supported by internal alpha-helices. The fact that these structural motifs are associated with the midline of the dimer indicates an importance concerning the formation of the dimer; possibly due to hydrophobic interactions of the alpha-helical side chains.
Another area of activity and structural importance is the region of DLDH that binds to the dihydrolipoyl transacetylase subunit (E2). This region has been called an electrostatic zipper and consists of Arg136 (Reset) (click on reset)
The subunit of DLDH has mainly been studied from the aspect of FAD being bound as a cofactor (only one monomer will be shown to ease visualization)
The FAD cofactor is within a domain of the protein that consists of a central parallel beta-sheet flanked on one face by three alpha-helices and on the other by an anti-parallel beta-sheet (the region described earlier).
The activity of DLDH in the reduction and subsequent oxidation of its cofactor, FAD, is strongly linked to the existance of 2 Cystein residues located in the active site as well as Histidine 446. The His446
The conformational change that takes place during the reduction of FAD to FADH2 causes a shift in the chain allowing a "tunnel" to form, by the moving of the Tyr186 side chain. This shift allows for the interaction of NAD+ and FADH2. Subsequently the FADH2 is oxidized to FAD and the NAD+ is reduced to NADH plus H+. The disulfide bridge between Cys47 and Cys52 is then reformed and the original E3 subunit is hence reconstituted.
Along with these important residues their seem to be others that are involved in the active site of the enzyme based on protein and DNA sequencing done on patients with DLDH deficiency. Some of these include Lys37, Pro453, and Arg460. The substitutions that were observed varied in side-chain charge and/or hydrophobicity.
The primary structure of an enzyme being changed can cause a shift in the shape of an enzyme. Even a single amino acid change in an enzyme can cause a change in the active site and therefore decrease efficiency, as medical findings of DLDH support. These minor changes in sequence have such an effect on the active site due to their interference with the natural noncovalent interactions in the wild type enzyme. These changes then cause a structural change that subsequently causes a functional change.