Cystathionine beta synthase

Cystathionine β-synthase (CBS) is a multidomainal enzyme with three modules and a heme domain. CBS catalyzes a pyridoxal-phosphate (PLP)-dependent β-replacement reaction condensing homocysteine and serine to form cystathionine and can be allosterically regulated by effectors such as the ubiquitous cofactor S-adenosyl-L-methionine (adoMet). The reaction catalyzed by CBS is the committed step in the synthesis of cysteine from methionine by transsulfuration and is the most common locus for mutations associated with homocystinuria^[1].

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Structure

The human enzyme cystathionine β-synthase is a tetramer and comprises 551 amino acids with a subunit molecular weight of 63 kDa. It displays a modular organization of three modules with the N-terminal heme domain followed by a core that contains the PLP cofactor^[2].The cofactor is deep in the heme domain and is linked by a Schiff base^[3]. A Schiff base is a functional group containing a C=N bond with the nitrogen atom connected to an aryl or alkyl group. The heme domain is composed of 70 amino acids and it appears that the heme only exists in mammalian CBS and is absent in yeast and protozoan CBS. At the C-terminus, the regulatory domain of CBS contains a tandem repeat of two CBS domains of β-α-β-β-α, a secondary structure motif found in other proteins^[2]. CBS has a C-terminal inhibitory domain. The C-terminal domain of cystathionine β-synthase regulates its activity via both intrasteric and allosteric effects and is important for maintaining the tetrameric state of the protein^[2]. This inhibition is alleviated by binding of the allosteric effector, adoMet, or by deletion of the regulatory domain; however, the magnitude of the effects differ^[2]. Mutations in this domain are correlated with hereditary diseases^[4].

The heme domain contains an N-terminal loop that binds heme and provides the axial ligands C52 and H65. The distance of heme from the PLP binding site suggests its non-role in catalysis, however deletion of the heme domain causes loss of redox sensitivity, therefore it is hypothesized that heme is a redox sensor^[3]. The presence of protoporphyrin IX in CBS is a unique PLP-dependent enzyme and is only found in the mammalian CBS. Drosophila melanogaster and Dictyostelium discoides have truncated N-terminal extensions and therefore prevent the conserved histidine and cysteine heme ligand residues. However, the Anopheles gambiae sequence has a longer N-terminal extension than the human enzyme and contains the conserved histidine and cysteine heme ligand residues like the human heme. Therefore, it is possible that CBS in slime molds and insects are hemeproteins that suggest that the heme domain is an early evolutionary innovation that arose before the separation of animals and the slime molds^[2]. The PLP is an internal aldimine and forms a Schiff base with K119 in the active site. Between the catalytic and regulatory domains exists a hypersensitive site that causes proteolytic cleavage and produces a truncated dimeric enzyme that is more active than the original enzyme. Both truncated enzyme and the enzyme found in yeast are not regulated by adoMet. The yeast enzyme is also activated by the deletion of the C-terminal to produce the dimeric enzyme^[2].

Enzymatic Activity

Transsulfuration, catalyzed by CBS, converts homocysteine to cysteine via a cystathionine intermediate^[5]. CBS occupies a pivotal position in mammalian sulfur metabolism at the homocysteine junction where the decision to conserve methionine or to convert it to cysteine via the transsulfuration pathway, is made. Moreover, the transsulfuration pathway is the only pathway capable of removing sulfur-containing amino acids under conditions of excess^[2].

In analogy with other β-replacement enzymes, the reaction catalyzed by CBS is predicted to involve a series of adoMet-bound intermediates. Addition of serine results in a transchiffization reaction, which forms of an external aldimine. The aldimine undergoes proton abstraction at the α-carbon followed by elimination to generate an aminoacrylate intermediate. Nucleophilic attack by the thiolate of homocysteine on the aminoacrylate and reprotonation at Cα generate the external aldimine of cystathionine. A final transaldimination reaction releases the final product, cystathionine^[2]. The final product, L-cystathionine can also form an aminoacrylate intermediate, indicating that the entire reaction of CBS is reversible^[6].

The measured V0 of an enzyme-catalyzed reaction generally reflects the steady state (where [ES] is constant), even though V0 is limited to the early part of a reaction, and analysis of these initial rates is referred to as steady-state kinetics. Steady-state kinetic analysis of yeast CBS yields parallel lines. These results agree with the proposed ping-pong mechanism in which serine binding and release of water are followed by homocysteine binding and release of cystathionine. In contrast, the steady-state enzyme kinetics of rat CBS yields intersecting lines, indicating that the β-substitutent of serine is not released from the enzyme prior to binding of homocysteine^[2].

One of the potential alternate reactions involving CBS is the condensation of cysteine with homocysteine to form cystathionine and hydrogen sulfide (H2S)^[6]. H2S in the brain is produced from L-cysteine by CBS. CBS and adoMet expressed in the brain enhance H2S production. This alternative metabolic pathway is also dependent on adoMet^[7].

CBS enzyme activity is not found in all tissues and cells. It is absent from heart, lung, testes, adrenal, and spleen in rats. In humans, it has been shown to be absent in heart muscle and primary cultures of human aortic endothelial cells. The lack of CBS in these tissues implies that these tissues are unable to synthesize cysteine and that cysteine must be supplied extracellularly. It also suggests that these tissues might have increased sensitivity to homocysteine toxicity because they cannot catabolize excess homocysteine via transsulfuration^[6].

Regulation

Allosteric activation of CBS adoMet determines the metabolic fate of L-homocysteine. Mammalian CBS is activated 2.5-5-fold by adoMet with a dissociation constant of 15 µM^[1]. adoMet is an allosteric activator that increases the Vmax of the CBS reaction but does not affect the Kms for the substrates. In other words, adoMet stimulates CBS activity by increasing the turnover rate rather than the binding of substrates to the enzyme^[2].

Human CBS performs a crucial step in the biosynthetic pathway of cysteine by providing a regulatory control point for adoMet . L-homocysteine, after being methylated to methionine, can be converted to adoMet , which donates methyl groups to a variety of substrates, e.g., neurotransmitters, proteins, and nucleic acids. adoMet functions as an allosteric activator of CBS and exerts control on its biosynthesis: low concentrations of adoMet result in low CBS activity, thereby funneling homocysteine in the transmethylation pathway toward adoMet formation. Methionine is transmethylated to homocysteine via S-adenosyl-methionine (SAM). In contrast, high adoMet concentrations allow the clearance of homocysteine into the transsulfuration pathway, leading to cysteine biosynthesis^[8].

In mammals, CBS is a highly regulated enzyme, which contains a heme cofactor that functions as a redox sensor^[9], that can modulate its activity in response to changes in the redox potential. If the resting form of CBS in the cell has ferrous heme, the potential exists for activating the enzyme under oxidizing conditions by conversion to the ferric state^[2]. The Fe (II) form of the enzyme is inhibited upon binding CO or nitric oxide, whereas enzyme activity is doubled when the Fe (II) is oxidized to Fe (III). The redox state of the heme is pH dependent, with oxidation of Fe (II)-CBS to Fe (III)-CBS being favored at low pH conditions^[10]. Since mammalian CBS contains a heme cofactor, whereas yeast and protozoan enzyme from Trypanosoma cruzi do not have heme cofactors, researchers have speculated that heme is not required for CBS activity^[2].

Human Disease

Hyperhomocysteinemia is a medical condition characterized by an abnormally large level of homocysteine in the blood. Mutations in CBS are the single most common cause of hereditary hyperhomocysteinemia. Inborn errors in CBS result in hyperhomocysteinemia with complications in the cardiovascular system leading to early and aggressive arterial disease. Hyperhomocysteinemia also affects three other major organ systems including the ocular, central nervous, and skeletal^[2].

Homocystinuria due to CBS deficiency is a special type of hyperhomocysteinemia. It is a rare, hereditary recessive autosomal disease generally diagnosed during childhood. A total of 131 different homocystinuria-causing mutations have been identified. A common functional feature of the mutations in the CBS domains is that the mutations abolish or strongly reduce activation by adoMet^[8].

Bioengineering

Researches have suggested that cystathionine beta synthase (CBS) is involved in oocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary and research is now focused on determining the location and expression during follicle development in the ovaries^[11].

• Cystathionine beta-synthase in BRENDA: The Comprehensive Enzyme Information System

• Wellcome Trust Sanger Institute Page on Cystathionine beta-synthase domain

• Cystathionine beta-Synthase: Protein Data Bank Entry

See also

Homocystinuria Cysteine Metabolism Amino Acids S-Adenosyl-L-methionine Heme

References