The molecular basis for specific lipid protein interactions in membranes remains unresolved although such selective interactions are manifest in or regulate a variety of biological processes. The goal of this research is to determine the molecular basis of the role for phospholipid(s) in the function of a well characterized lipid requiring enzyme, (R) 3 hydroxybutyrate dehydrogenase (BDH), that specifically requires phosphatidylcholine (PC) for function. BDH is a mitochondrial membrane enzyme which interconverts (R) 3 hydroxybutyrate and acetoacetate, metabolites that are particularly important both in neonatal development and in diabetes. Purified BDH, devoid of lipids and detergents (apoBDH), is inactive but activity can be restored by reconstitution with membranes or phospholipid vesicles containing PC. The activation of BDH by PC is cooperative and PC serves as an allosteric activator to enhance the binding affinity of BDH for coenzyme, NAD(H), by ca. 50 fold. Recently, the cDNA encoding human heart (HH) BDH has been cloned and the enzyme expressed in functional form so that new approaches to study lipid BDH molecular interaction are now practical. HH BDH and specific variants, prepared by site directed mutagenesis, will be characterized using biochemical and biophysical methods to address the molecular basis for the PC requirement.
The specific aims are to: 1) complete the purification and characterization of HH BDH expressed in insect cells; 2) test models for the predicted domain structure of BDH and to identify specific amino acids and/or parts of the sequence important for lipid binding and/or catalytic function of the enzyme; 3) assess the role of cysteine in the function of HH BDH; and 4) determine the topography of specific sites of BDH with respect to the phospho- lipid bilayer.
For Aims 1 to 3, cloned and expressed HH BDH and variants will be purified, reconstituted into phospholipid vesicles and characterized. Specific residues or segments of BDH sequence will be selected (based on known chemistry and/or sequence homology) and modified by site directed mutagenesis to evaluate their role in enzymic function (e.g., catalysis, PC requirement for function, ligand binding characteristics, etc.).
For Aim 4, fluorescence quenching or energy transfer studies will be used to determine the location of specific sites on BDH [e.g., NAD(H) site or selectively labelled sites introduced by mutagenesis (""""""""site directed labelling"""""""")] with respect to probes (spin label, bromine or BODIPY) located at specific sites in the phospholipid bilayer. Results from these studies should elucidate the molecular basis of the allosteric lipid requirement for BDH function and thereby provide new information about specific lipid protein interactions in membranes.
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| Loeb-Hennard, C; McIntyre, J O (2000) (R)-3-hydroxybutyrate dehydrogenase: selective phosphatidylcholine binding by the C-terminal domain. Biochemistry 39:11928-38 |
| Picklo, M J; Amarnath, V; McIntyre, J O et al. (1999) 4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple sites. J Neurochem 72:1617-24 |
