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Structural basis of allosteric and intrasteric regulation in human cystathionine β-synthase and its regulation by a CXXC motif
Abstract
The human cystathionine β-synthase (CBS) is a 5' pyridoxal phosphate (PLP) dependent protein that catalyzes the condensation reaction between serine and homocysteine to yield cystathionine. This is the first step in the transsulfuration pathway which connects the methionine cycle to cysteine production. Mutations in CBS are the single most common cause of severe hereditary hyperhomocystenemia and over one hundred pathogenic mutations have been described so far. These mutations represent residues which are important for the structural and functional integrity of the enzyme. The biochemical characterization of these mutants thus leads to further insights into structure-function correlations in CBS. CBS has a subunit mass of 63 kDa and is a modular protein with 551 amino acids. The N-terminal half of the protein houses the two cofactors, PLP and heme and a CXXC motif comprised of residues C272 P273, G274 and C275. This motif is also found in the thioredoxin family of proteins where it is involved in thiol disulfide exchange reactions. The heme, which is coordinated by the axial ligands H65 and C52, is about 20 Å away from the CXXC motif as well as the PLP catalytic center. As such, heme does not contribute directly to catalysis, but is believed to be a center for redox regulation in CBS. The C-terminal half of the protein comprising residues 413 to 551 bear a tandem repeat of “CBS domains,” which are secondary structural motifs found in diverse families of proteins with no sequence similarities. In CBS, these domains are believed to be involved in the binding of the allosteric activator S-adenosyl methionine (AdoMet). Along with being involved in allosteric regulation, the C-terminal domain of CBS imposes intrasteric regulation on the catalytic core. Thus deletion of the C-terminal regulatory domain leads to the formation of a super-active enzyme, CBS-ΔC143 which has a 4-fold higher kcat than the full-length protein. It is also known that a truncated form of CBS is generated in cells exposed to proinflammatory agents such as TNFα. Although this observation suggests that the C-terminal domain interacts with the N-terminal catalytic core to effect this regulation, so far no experimental results were available which would characterize the nature of this interaction. In other words no information was available regarding the conformational changes associated with the allosteric and intrasteric regulation in CBS. In this study, we have mapped the regions of intrasteric and allosteric regulation in CBS. We have employed hydrogen/deuterium (H/D) exchange, mass spectrometric technique to locate conformational changes in the enzyme upon the binding of its allosteric activator, AdoMet. We have also used this approach to detect the surface involved in the interaction of the C-terminal domain with the catalytic core relevant to intrasteric regulation. A change in the kinetics of H/D exchange was located in a single peptide, extending from residues 511–531 upon AdoMet binding. CBS-D444N, a patient mutant, exhibits a similar change in the above peptide in its native state and thus samples a conformation which is acquired by the wild type upon AdoMet binding. Accordingly this mutant is not responsive to any further activation by the allosteric activator. Peptides 356–370 and 371–385 in the N-terminal half of the protein exhibited a change in H/D exchange kinetics when the full-length and its counter part in CBS-ΔC143, were compared by H/D exchange studies, associating this region with intrasteric regulation. We have also demonstrated that in addition to heme, the CXXC motif in CBS is a center for redox regulation. Thiol alkylation followed by mass spectrometric analysis demonstrated formation of an intramolecular disulfide bridge between C272 and C275, and identified the presence of a sulfenic acid intermediate under air oxidized conditions in CBS-ΔC143. The full length and the CBS-ΔC143 enzyme exhibited a 1.6 and 4.5 fold enhancement in specific activity respectively upon reduction of their disulfides at the CXXC center. A redox potential of −240 ± 4 mV was determined for the CXXC center using MAL-PEG for titrating thiol content at various ratios of oxidized and reduced dithiotreitol.
Subject Area
Biochemistry
Recommended Citation
Sen, Suvajit, "Structural basis of allosteric and intrasteric regulation in human cystathionine β-synthase and its regulation by a CXXC motif" (2007). ETD collection for University of Nebraska-Lincoln. AAI3271912.
https://digitalcommons.unl.edu/dissertations/AAI3271912