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Role of heme in the reaction catalyzed by human cystathionine beta-synthase

Sunil Ojha, University of Nebraska - Lincoln

Abstract

CBS binds two cofactors, heme and PLP, which catalyze diverse reactions. The role of PLP in the CBS-catalyzed reaction is to activate serine, a substrate, to eliminate the hydroxyl group and to facilitate the nucleophilic attack by homocysteine. The role of heme is not known. Studies done in our laboratory indicate that the role of the heme is to regulate enzyme activity. ^ We have utilized chemical mutagenesis to identify the heme ligands. Binding of HgCl2, a thiol chelator, perturbs the UV-visible and the EPR spectrum which indicates that the low-spin heme converts to the high-spin. These data indicate that one of the axial ligands of CBS is thiolate of cysteine. Recently, the structure of the active site core of the enzyme has been solved and H65 and C52 have been identified as heme axial ligands. We have made a conservative mutation and nonconservative mutation of these heme ligands. These mutants show lower heme saturation and weaker PLP binding. Despite the full complement of PLP, these mutants retain only ∼20% of the wild type activity. The pH profile of these mutants is significantly altered compared to the wild type enzyme. ^ These data indicate that changes in the heme binding domain can alter the activity of the enzyme. We have deleted the entire heme binding domain comprising the 69 amino acid residues from the N-terminus of the human CBS. The addition of serine and cystathionine to the heme domain deleted mutant (CBS-NΔ69) forms predominantly the external aldimine. S-Adenosyl methionine (AdoMet) is an allosteric activator of human CBS and its binding activated the enzyme ∼2-fold. Deletion of the heme domain of human enzyme reverses the AdoMet response. AdoMet binding to CBS-NΔ69 inhibits the activity by ∼1.5-fold. These data indicates that interactions between the C-terminal and N-terminal domain are important in the architecture of the functional enzyme. ^

Subject Area

Biology, Molecular|Chemistry, Biochemistry

Recommended Citation

Ojha, Sunil, "Role of heme in the reaction catalyzed by human cystathionine beta-synthase" (2002). ETD collection for University of Nebraska - Lincoln. AAI3074091.
http://digitalcommons.unl.edu/dissertations/AAI3074091

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