Heme Regulation of Human Cystathionine β-Synthase Activity: Insights from Fluorescence and Raman Spectroscopy
- 10 August 2009
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 131 (35), 12809-12816
- https://doi.org/10.1021/ja904468w
Abstract
Cystathionine β-synthase (CBS) plays a central role in homocysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5′-phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift of the PLP from the ketoenamine to the enolimine form. The ketoenamine is key to PLP reactivity because its imine C═N bond is protonated, facilitating attack by the nucleophilic substrate, serine. The same tautomer shift is also induced by heat inactivation of Fe(II)CBS, or by an Arg266Met replacement in Fe(II)CBS, which likewise inactivates the enzyme; in both cases the endogenous Cys52 ligand to the heme is replaced by another, unidentified ligand. CO binding also displaces Cys52 from the heme. We propose that the tautomer shift results from loss of a stabilizing H-bond from Asn149 to the PLP ring O3′ atom, which is negatively charged in the ketoenamine tautomer. This loss would be induced by displacement of the PLP as a result of breaking the salt bridge between Cys52 and Arg266, which resides on a short helix that is also anchored to the PLP via H-bonds to its phosphate group. The salt bridge would be broken when Cys52 is displaced from the heme. Cys52 protonation is inferred to be the rate-limiting step in breaking the salt bridge, since the rate of the tautomer shift, following CO binding, increases with decreasing pH. In addition, elevation of the concentration of phosphate buffer was found to diminish the rate and extent of the tautomer shift, suggesting a ketoenamine-stabilizing phosphate binding site, possibly at the protonated imine bond of the PLP. Implications of these findings for CBS regulation are discussed.This publication has 64 references indexed in Scilit:
- Modulation of the heme electronic structure and cystathionine β-synthase activity by second coordination sphere ligands: The role of heme ligand switching in redox regulationJournal of Inorganic Biochemistry, 2009
- Active Cystathionine β-Synthase Can Be Expressed in Heme-free Systems in the Presence of Metal-substituted Porphyrins or a Chemical ChaperoneOnline Journal of Public Health Informatics, 2008
- H 2 S as a Physiologic Vasorelaxant: Hypertension in Mice with Deletion of Cystathionine γ-LyaseScience, 2008
- Cystathionine β-synthase as a carbon monoxide-sensitive regulator of bile excretionHepatology, 2008
- Hydrogen sulfide: Neurochemistry and neurobiologyNeurochemistry International, 2008
- The Heme of Cystathionine β-synthase Likely Undergoes a Thermally Induced Redox-Mediated Ligand SwitchBiochemistry, 2005
- A Novel Enhancing Mechanism for Hydrogen Sulfide-producing Activity of Cystathionine β-SynthaseOnline Journal of Public Health Informatics, 2002
- Assignment of Enzymatic Functions to Specific Regions of the PLP-Dependent Heme Protein Cystathionine β-SynthaseBiochemistry, 1999
- Ionisation of Cysteine Residues at the Termini of Model α-Helical Peptides. Relevance to Unusual Thiol pKaValues in Proteins of the Thioredoxin FamilyJournal of Molecular Biology, 1995
- Equilibria between Pyridoxal and Amino Acids and their Imines1Journal of the American Chemical Society, 1957