Homocysteine stimulates vascular smooth muscle and endothelial nitric oxide (NO) production and impairs NO anticoagulant and vasodilatory functions, raising he possibility of a feed back loop, whereby NO regulates the levels of homocysteine through cystathionine beta-synthase (CBS). Dysfunction in this putative feedback loop may constitute a novel mechanism underlying certain types of thromboembolic disease. The studies described herein will provide insight into the role of heme in homocysteine metabolism, and reveal whether there is heme-related cross-talk between the trans-sulfuration and NO/cGMP pathways. Our studies will focus on CBS, a heme-containing enzyme that is critical to the control of physiological homocysteine levels. There are two overarching goals of this sub-project within the collaborative effort described in the accompanying proposal from Jan P. Kraus entitled """"""""Homocysteine and Thrombosis""""""""> The goal of this project are: 1) to elucidate the role of heme in CBS, and 2) to determine whether there is interaction between homocysteine and NO in vascular pathology. The biochemical studies described herein will be complemented by in vivo studies carried out in the Kraus laboratory. The primary hypothesis of this proposal is that heme in CBS is serving a regulatory function, similar to the roles now attributed to the heme moiety in the NO receptor, soluble guanylyl cyclase. We hypothesize that the heme is regulatory and that NO, carbon monoxide or another physiologically relevant is the regulator in vivo. The goal of the research proposed herein is to test this hypothesis. Our laboratory will test the biochemical feasibility, focusing primarily on characterizing the purified CBS protein and its interaction with exogenous ligands through spectroscopic and enzymological studies. The Kraus laboratory will explore the physiological feasibility through studies in tissue culture and knockout mouse models. There are three Specific Aims that we will address: 1) we will determine CBS is regulated by No or other heme ligands by correlating changes in the heme absorption spectrum on ligand binding with changes in enzyme activity, 2) we will identify structural changes at the CBS heme site relevant to enzyme function, characterizing the active core heme site and its ligand adducts by EPR and MCD spectroscopy, 3) we will apply the methods of Aim 1 and 2 to identify differences between the CBS active core and the full length polypeptide, correlating regulation by heme and regulation by Adomet.
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