Structure of human cystathionine beta-synthase: a unique pyridoxal 5'-phosphate-dependent heme protein

Cystathionine beta-synthase (CBS) is a unique heme- containing enzyme that catalyzes a pyridoxal 5'-phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine. Deficiency of CBS leads to homocystinuria, an inherited disease of sulfur metabolism characterized by increased levels of the toxic metabolite homocysteine. Here we present the X-ray crystal structure of a truncated form of the enzyme. CBS shares the same fold with O-acetylserine sulfhydrylase but it contains an additional N-terminal heme binding site. This heme binding motif together with a spatially adjacent oxidoreductase active site motif could explain the regulation of its enzyme activity by redox changes.     

Reaction mechanism and regulation of cystathionine beta-synthase

In mammals, cystathionine beta-synthase catalyzes the first step in the transsulfuration pathway which provides an avenue for the conversion of the essential amino acid, methionine, to cysteine. Cystathionine beta-synthase catalyzes a PLP-dependent condensation of serine and homocysteine to cystathionine and is unique in also having a heme cofactor. In this review, recent advances in our understanding of the kinetic mechanism of the yeast and human enzymes as well as pathogenic mutants of the human enzyme and insights into the role of heme in redox sensing are discussed from the perspective of the crystal structure of the catalytic core of the human enzyme.     

Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein

Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.     

Functional properties of the active core of human cystathionine beta-synthase crystals

Human cystathionine beta-synthase is a pyridoxal 5'-phosphate enzyme containing a heme binding domain and an S-adenosyl-l-methionine regulatory site. We have investigated by single crystal microspectrophotometry the functional properties of a mutant lacking the S-adenosylmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are reversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, which takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5'-phosphate-dependent protein. The exposure of these crystals to increasing concentrations of the natural substrate l-serine readily led to the formation of the key catalytic intermediate alpha-aminoacrylate. The dissociation constant of l-serine was found to be 6 mm, close to that determined in solution. The amount of the alpha-aminoacrylate Schiff base formed in the presence of l-serine was pH independent between 6 and 9. However, the rate of the disappearance of the alpha-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the alpha-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine beta-synthase crystals catalyze both beta-elimination and beta-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine beta-synthase.     

Characterization of the heme and pyridoxal phosphate cofactors of human cystathionine beta-synthase reveals nonequivalent active sites

Cystathionine beta-synthase is an unusual enzyme that requires the cofactors heme and pyridoxal phosphate (PLP) to catalyze the condensation of homocysteine and serine to generate cystathionine. This transsulfuration reaction represents one of two major cellular routes for detoxification of homocysteine, which is a risk factor for atherosclerosis. While the beta-replacement reaction catalyzed by this enzyme suggests a role for the pyridoxal phosphate, the role of the heme is uncertain. In this study we have examined the effect of changing one of the ligands to the heme on the activity of the enzyme. Binding of carbon monooxide results in the displacement of a thiolate ligand to the ferrous heme, and is accompanied by complete loss of cystathionine beta-synthase activity. Furthermore, inhibition by CO is competitive with respect to homocysteine, providing the first indication that the homocysteine binding site is in the proximity of heme. Binding of both CO and cyanide to ferrous cystathionine beta-synthase occurs in two distinct isotherms and indicates that the hemes are nonequivalent. We have employed fluorescence spectroscopy to characterize the bound PLP and its interaction with serine. PLP bound to cystathionine beta-synthase is weakly fluorescent and exists as a mixture of the protonated and unprotonated tautomers. Reaction with hydroxylamine releases the oxime and greatly enhances the associated fluorescence. Binding of serine is accompanied by a shift to the unprotonated tautomer of the external aldimine as well as the appearance of a new fluorescent species at approximately 400 nm that could be due to the aminoacrylate or to a gemdiamine intermediate. These data provide the first characterization of the PLP bound to cystathionine beta-synthase. Treatment of cystathionine beta-synthase with hydroxylamine releases two PLPs after 1 day and results in complete loss of activity. Incubation for an additional 3-4 days results in the release of two more PLPs. These data lead us to revise the PLP stoichiometry to 4 per tetramer, and to the conclusion that the heme and PLP sites in cystathionine beta-synthase are nonequivalent.     

Characterization of the heme in human cystathionine beta-synthase by X-ray absorption and electron paramagnetic resonance spectroscopies

Human cystathionine beta-synthase is one of two key enzymes involved in intracellular metabolism of homocysteine. It catalyzes a beta-replacement reaction in which the thiolate of homocysteine replaces the hydroxyl group of serine to give the product, cystathionine. The enzyme is unusual in its dependence on two cofactors: pyridoxal phosphate and heme. The requirement for pyridoxal phosphate is expected on the basis of the nature of the condensation reaction that is catalyzed; however the function of the heme in this protein is unknown. We have examined the spectroscopic properties of the heme in order to assign the axial ligands provided by the protein. The heme Soret peak of ferric cystathionine beta-synthase is at 428 nm and shifts to approximately 395 nm upon addition of the thiol chelator, mercuric chloride. This is indicative of 6-coordinate low-spin heme converting to a 5-coordinate high-spin heme. The enzyme as isolated exhibits a rhombic EPR signal with g values of 2.5, 2.3, and 1.86, which are similar to those of heme proteins and model complexes with imidazole/thiolate ligands. Mercuric chloride treatment of the enzyme results in conversion of the rhombic EPR signal to a g = 6 signal, consistent with formation of the high-spin ferric heme. The X-ray absorption data reveal that iron in ferric cystathionine beta-synthase is 6-coordinate, with 1 high-Z scatterer and 5 low-Z scatterers. This is consistent with the presence of 5 nitrogens and 1 sulfur ligand. Together, these data support assignment of the axial ligands as cysteinate and imidazole in ferric cystathionine beta-synthase.     

Human cystathionine beta-synthase is a heme sensor protein. Evidence that the redox sensor is heme and not the vicinal cysteines in the CXXC motif seen in the crystal structure of the truncated enzyme

Elevated levels of homocysteine, a sulfur-containing amino acid, are correlated with increased risk for cardiovascular diseases and Alzheimers disease and with neural tube defects. The only route for the catabolic removal of homocysteine in mammals begins with the pyridoxal phosphate- (PLP-) dependent beta-replacement reaction catalyzed by cystathionine beta-synthase. The enzyme has a b-type heme with unusual spectroscopic properties but as yet unknown function. The human enzyme has a modular organization and can be cleaved into an N-terminal catalytic core, which retains both the heme and PLP-binding sites and is highly active, and a C-terminal regulatory domain, where the allosteric activator S-adenosylmethionine is presumed to bind. Studies with the isolated recombinant enzyme and in transformed human liver cells indicate that the enzyme is approximately 2-fold more active under oxidizing conditions. In addition to heme, the enzyme contains a CXXC oxidoreductase motif that could, in principle, be involved in redox sensing. In this study, we have examined the role of heme versus the vicinal thiols in modulating the redox responsiveness of the enzyme. Deletion of the heme domain leads to loss of redox sensitivity. In contrast, substitution of either cysteine with a non-redox-active amino acid does not affect the responsiveness of the enzyme to reductants. We also report the crystal structure of the catalytic core of the enzyme in which the vicinal cysteines are reduced without any discernible differences in the remainder of the protein. The structure of the catalytic core is compared to those of other members of the fold II family of PLP-dependent enzymes and provides insights into active site residues that may be important in interacting with the substrates and intermediates.     

Visualization of PLP-bound intermediates in hemeless variants of human cystathionine beta-synthase: evidence that lysine 119 is a general base

Cystathionine beta-synthase catalyzes the condensation of serine and homocysteine to give cystathionine in a pyridoxal phosphate (PLP)-dependent reaction. The human enzyme contains a single heme per monomer that is bound in an N-terminal 69 amino acid extension that is missing from the otherwise highly homologous yeast enzyme. The heme dominates the UV-visible spectrum and obscures kinetic characterization of the PLP-bound reaction intermediates. In this study, we have engineered a hemeless mutant of human cystathionine beta-synthase by deletion of the N-terminal 69 amino acids. The resulting variant displays approximately 40% of the activity seen with the wild type enzyme, binds stoichiometric amounts of PLP, and permits spectral characterization of PLP-based intermediates. The enzyme as isolated exhibits an absorption maximum at 412nm corresponding to a protonated internal aldimine. Addition of serine shifts the lambdamax to 420nm (assigned as the external aldimine) with a broad shoulder between 450 and 500nm (assigned as the aminoacrylate intermediate). Addition of the product, cystathionine, also leads to formation of an external aldimine (420nm). Homocysteine elicits a red shift (and a decrease in absorption) in the spectrum from 412 to 424nm and an increase in absorption at 330nm, presumably due to formation of a dead-end complex. Mutation of K119, the residue that forms the Schiff base, to alanine results in a approximately 10(3)-fold decrease in activity, which increases approximately 2-fold in the presence of an exogenous base, ethylamine. Spectral shifts (412 --> 420nm) consistent with the formation of external aldimines are observed in the presence of serine or cystathionine, but an aminoacrylate intermediate is not formed at detectable levels. These results are consistent with an additional role for K119 as a general base in the reaction catalyzed by human cystathionine beta-synthase.     

Modulation of cystathionine beta-synthase activity by the Arg-51 and Arg-224 mutations

Human cystathionine beta-synthase (CBS) catalyzes a pyridoxal 5'-phosphate (PLP) dependent beta-replacement reaction to synthesize cystathionine from serine and homocysteine. The enzyme is unique in bearing not only a catalytically important PLP but also heme. In order to study a regulatory process mediated by heme, we performed mutagenesis of Arg-51 and Arg-224, which have hydrogen-bonding interactions with propionate side chains of the prosthetic group. It was found that the arginine mutations decrease CBS activity by approximately 50%. The results indicate that structural changes in the heme vicinity are transmitted to PLP existing 20 A away from heme. A possible explanation of our results is discussed on the basis of CBS structure.     

Alleviation of intrasteric inhibition by the pathogenic activation domain mutation,D444N,in human cystathionine beta-synthase

Human cystathionine beta-synthase is a heme protein that catalyzes the condensation of serine and homocysteine to form cystathionine in a pyridoxal phosphate-dependent reaction. Mutations in this enzyme are the leading cause of hereditary hyperhomocysteinemia with attendant cardiovascular and other complications. The enzyme is activated approximately 2-fold by the allosteric regulator S-adenosylmethionine (AdoMet), which is presumed to bind to the C-terminal regulatory domain. The regulatory domain exerts an inhibitory effect on the enzyme, and its deletion is correlated with a 2-fold increase in catalytic activity and loss of responsiveness to AdoMet. A mutation in the C-terminal regulatory domain, D444N, displays high levels of enzyme activity, yet is pathogenic. In this study, we have characterized the biochemical penalties associated with this mutation and demonstrate that it is associated with a 4-fold lower steady-state level of cystathionine beta-synthase in a fibroblast cell line that is homozygous for the D444N mutation. The activity of the recombinant D444N enzyme mimics the activity of the wild-type enzyme seen in the presence of AdoMet and can be further activated approximately 2-fold in the presence of supraphysiolgical concentrations of the allosteric regulator. The mutation increases the K(act) for AdoMet from 7.4 +/- 0.2 to 460 +/- 130 microM, thus rendering the enzyme functionally unresponsive to AdoMet under physiological concentrations. These results indicate that the D444N mutation partially abrogates the intrasteric inhibition imposed by the C-terminal domain. We propose a model that takes into account the three kinetically distinguishable states that are observed with human cystathionine beta-synthase: "basal" (i.e., wild-type enzyme as isolated), "activated" (wild-type enzyme + AdoMet or the D444N mutant as isolated), and superactivated (D444N mutant + AdoMet or wild-type enzyme lacking the C-terminal regulatory domain).     

Deletion of the regulatory domain in the pyridoxal phosphate-dependent heme protein cystathionine beta-synthase alleviates the defect observed in a catalytic site mutant.

The most common cause of severely elevated homocysteine or homocystinuria is inherited disorders in cystathionine beta-synthase. The latter enzyme is a unique hemeprotein that catalyzes pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine, thus committing homocysteine to catabolism. A point mutation, V168M, has been described in a homocystinuric cell line and is associated with a B(6)-responsive phenotype. In this study, we have examined the kinetic properties of this mutant and demonstrate that the mutation affects the PLP but not the heme content. The approximately 13-fold diminution in activity because of the mutation corresponds to an approximately 7-fold decrease in the level of bound PLP. This may be explained by half of the sites activity associated with cystathionine beta-synthase. The addition of PLP results in partial but not full restoration of activity to wild type levels. Elimination of the C-terminal quarter of the mutant protein results in alleviation of the catalytic penalty imposed by the V168M mutation. The resulting truncated protein is very similar to the corresponding truncated enzyme with wild type sequence and is now able to bind the full complement of both heme and PLP cofactors. These results indicate that the V168M mutation per se does not affect binding of PLP directly and that interactions between the regulatory C terminus and the catalytic N terminus are important in modulating the cofactor content and therefore the activity of the full-length enzyme. These studies provide the first biochemical explanation for the B(6)-responsive phenotype associated with a cystathionine beta-synthase-impaired homocystinuric genotype.     

Dioxygen reactivity and heme redox potential of truncated human cystathionine beta-synthase

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 +/- 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 +/- 0.07) x 10 (5) M (-1) s (-1) at pH 7.4 and 25 degrees C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical.     

Stopped-flow kinetic analysis of the reaction catalyzed by the full-length yeast cystathionine beta-synthase

Cystathionine beta-synthase found in yeast catalyzes a pyridoxal phosphate-dependent condensation of homocysteine and serine to form cystathionine. Unlike the homologous mammalian enzymes, yeast cystathionine beta-synthase lacks a second cofactor, heme, which facilitates detailed kinetic studies of the enzyme because the different pyridoxal phosphate-bound intermediates can be followed by their characteristic absorption spectra. We conducted a rapid reaction kinetic analysis of the full-length yeast enzyme in the forward and reverse directions. In the forward direction, we observed formation of the external aldimine of serine (14 mm(-1) s(-1)) and the aminoacrylate intermediate (15 s(-1)). Homocysteine binds to the aminoacrylate with a bimolecular rate constant of 35 mm(-1) s(-1) and rapidly converts to cystathionine (180 s(-1)), leading to the accumulation of a 420 nm absorbing species, which has been assigned as the external aldimine of cystathionine. Release of cystathionine is slow (k = 2.3 s(-1)), which is similar to k(cat) (1.7 s(-1)) at 15 degrees C, consistent with this being a rate-determining step. In the reverse direction, cystathionine binds to the enzyme with a bimolecular rate constant of 1.5 mm(-1) s(-1) and is rapidly converted to the aminoacrylate without accumulation of the external aldimine. The kinetic behavior of the full-length enzyme shows notable differences from that reported for a truncated form of the enzyme lacking the C-terminal third of the protein (Jhee, K. H., Niks, D., McPhie, P., Dunn, M. F., and Miles, E. W. (2001) Biochemistry 40, 10873-10880).     

Resonance Raman characterization of the heme cofactor in cystathionine beta-synthase. Identification of the Fe-S(Cys) vibration in the six-coordinate low-spin heme

Human cystathionine beta-synthase (CBS) is an essential enzyme for the removal of the toxic metabolite homocysteine. Heme and pyridoxal phosphate (PLP) cofactors are necessary to catalyze the condensation of homocysteine and serine to generate cystathionine. While the role for the PLP cofactor is thought to be similar to that in other PLP-dependent enzymes that catalyze beta-replacement reactions, the exact role for the heme remains unclear. In this study, we have characterized the heme cofactor of CBS in both the ferric and ferrous states using resonance Raman spectroscopy. Positive identification of a cysteine ligand was achieved by global (34)S isotopic substitution which allowed us to assign the nu(Fe-S) for the six-coordinate low-spin ferric heme at 312 cm(-1). In addition, the CO adduct of ferrous CBS has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment, and indicates that the Fe-S(Cys) bond is labile. We have also found that addition of HgCl(2) to the ferric heme causes conversion of the low-spin heme to a five-coordinate high-spin heme with loss of the cysteine ligand. The present spectroscopic studies do not support a reaction mechanism in which homocysteine binds directly to the heme via displacement of the Cys ligand in the binary enzyme complex, as had been previously proposed.     

Effects of heme ligand mutations including a pathogenic variant, H65R, on the properties of human cystathionine beta-synthase

Human cystathionine beta-synthase is a hemeprotein that catalyzes a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine into cystathionine. Biophysical characterization of this enzyme has led to the assignment of the heme ligands as histidine and cysteinate, respectively, which has recently been confirmed by crystal structure determination of the catalytic core of the protein. Using site-directed mutagenesis, we confirm that C52 and H65 represent the thiolate and histidine ligands to the heme. Conversion of C52 to alanine or serine results in spectral properties of the resulting hemeprotein that are consistent with the loss of a thiolate ligand. Thus, the Soret peak blue-shifts from 428 to 415 and 417 nm in the ferric forms of the C52S and C52A mutants, respectively, and from 450 to 423 nm in the ferrous states of both mutants. Addition of CO to the dithionite-reduced ferrous C52 mutants results in spectra with Soret peaks at 420 nm. EPR spectroscopy of the ferric C52 variants reveals the predominance of a high-spin species. The H65R mutant, a variant described in a homocystinuric patient, has Soret peaks at 424, 421, and 420 nm in the ferric, ferrous, and ferrous CO states, respectively. EPR spectroscopy reveals predominance of the low-spin species. Both C52A and C52S mutations lead to protein with substoichiometric heme (19% with respect to wild type); however, the PLP content is comparable to that of wild-type enzyme. The heme and PLP contents of the H65R mutant are 40% and 75% that of wild-type enzyme. These results indicate that heme saturation does not dictate PLP saturation in these mutant enzymes. Both H65 and C52 variants display low catalytic activity, revealing that changes in the heme binding domain modulate activity, consistent with a regulatory role for this cofactor.     

The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes

Homocysteine is a key junction metabolite in methionine metabolism. It suffers two major metabolic fates: transmethylation catalyzed by methionine synthase or betaine homocysteine methyl transferase and transsulfuration catalyzed by cystathionine beta-synthase leading to cystathionine. The latter is subsequently converted to cysteine, a precursor of glutathione. Studies with purified mammalian methionine synthase and cystathionine beta-synthase have revealed the oxidative sensitivity of both junction enzymes, suggesting the hypothesis that redox regulation of this pathway may be physiologically significant. This hypothesis has been tested in a human hepatoma cell line in culture in which the flux of homocysteine through transsulfuration under normoxic and oxidative conditions has been examined. Addition of 100 microM H(2)O(2) or tertiary butyl hydroperoxide increased cystathionine production 1.6- and 2.1-fold from 82 +/- 7 micromol h(-)(1) (L of cells)(-)(1) to 136 +/- 15 and 172 +/- 23 micromol h(-)(1) (L of cells)(-)(1), respectively. The increase in homocysteine flux through the transsulfuration pathway exhibited a linear dose dependence on the concentrations of both oxidants (50-200 microM H(2)O(2) and 10-200 microM tertiary butyl hydroperoxide). Furthermore, our results reveal that approximately half of the intracellular glutathione pool in human liver cells is derived from homocysteine via the transsulfuration pathway. The redox sensitivity of the transsulfuration pathway can be rationalized as an autocorrective response that leads to an increased level of glutathione synthesis in cells challenged by oxidative stress. In summary, this study demonstrates the importance of the homocysteine-dependent transsulfuration pathway in the maintenance of the intracellular glutathione pool, and the regulation of this pathway under oxidative stress conditions. Aberrations in this pathway could compromise the redox buffering capacity of cells, which may in turn be related to the pathophysiology of the different homocysteine-related diseases.     

Binding of pyridoxal 5'-phosphate to the heme protein human cystathionine beta-synthase

Cystathionine beta-synthase (CBS), a pyridoxal 5'-phosphate (PLP) dependent enzyme, catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS was recently shown to be a heme protein. While the role of heme in CBS is unknown, catalysis by CBS can be explained solely by participation of PLP in the reaction mechanism. In this study, treatment of CBS with sodium borohydride selectively reduced the Schiff base but did not affect the heme. Purification and sequencing of the PLP-cross-linked peptide from a trypsin digest of the reduced enzyme revealed the evolutionarily conserved Lys119 to be the residue forming the Schiff base. Serine and hydroxylamine form an alpha-aminoacrylate and an oxime with PLP in CBS, respectively. The sulfhydryl-containing substrate, homocysteine, disturbs the heme environment but does not interact with PLP. In contrast to other PLP-dependent enzymes, CBS emits no PLP-related fluorescence when excited at 296 or 330 nm. PLP but not heme dissociates from the enzyme in the presence of hydroxylamine. The dissociation of PLP is a multistage process involving a short approximately 500 s lag phase, followed by a rapid inactivation and a slower PLP-oxime formation. PLP-free CBS exhibits a decrease of secondary structure as well as loss of CBS activity that can be only partially restored by PLP. This study constitutes the first comprehensive investigation of PLP interaction with a heme protein.     

Ferrous human cystathionine beta-synthase loses activity during enzyme assay due to a ligand switch process

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS also contains a heme cofactor that has been proposed to allosterically regulate enzyme activity via the heme redox state, with FeII CBS displaying approximately half the activity of FeIII CBS in vitro. The results of this study show that human FeII CBS spontaneously loses enzyme activity over the course of a 20 min enzyme assay. Both the full-length 63-kDa and truncated 45-kDa form of CBS slowly and irreversibly lose activity upon reduction to the FeII form. Additionally, electronic absorption spectroscopy reveals that FeII CBS undergoes a heme ligand exchange to FeII CBS424 when the enzyme is incubated at 37 degrees C and pH 8.6. The addition of enzyme substrates or imidazole has a moderate effect on the rate of the ligand switch, but does not prevent conversion to the inactive species. Time-dependent spectroscopic data describing the conversion of FeII CBS to FeII CBS424 were fitted to a three-state kinetic model. The resultant rate constants were used to fit assay data and to estimate the activity of FeII CBS prior to the ligand switch. Based on this fit it appears that FeII CBS initially has the same enzyme activity as FeIII CBS, but FeII CBS loses activity as the ligand switch proceeds. The slow and irreversible loss of FeII CBS enzyme activity in vitro resembles protein denaturation, and suggests that a simple regulatory mechanism based on the heme redox state is unlikely.     

The redox behavior of the heme in cystathionine beta-synthase is sensitive to pH

Human cystathionine beta-synthase (CBS) is a unique pyridoxal-5'-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated.     

Domain architecture of the heme-independent yeast cystathionine beta-synthase provides insights into mechanisms of catalysis and regulation

Cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) provides a model system for understanding some of the effects of disease-causing mutations in the human enzyme. The mutations, which lead to accumulation of L-homocysteine, are linked to homocystinuria and cardiovascular diseases. Here we characterize the domain architecture of the heme-independent yeast cystathionine beta-synthase. Our finding that the homogeneous recombinant truncated enzyme (residues 1-353) is catalytically active and binds pyridoxal phosphate stoichiometrically establishes that the N-terminal residues 1-353 compose a catalytic domain. Removal of the C-terminal residues 354-507 increases the specific activity and alters the steady-state kinetic parameters including the K(d) for pyridoxal phosphate, suggesting that the C-terminal residues 354-507 compose a regulatory domain. The yeast enzyme, unlike the human enzyme, is not activated by S-adenosyl-L-methionine. The truncated yeast enzyme is a dimer, whereas the full-length enzyme is a mixture of tetramer and octamer, suggesting that the C-terminal domain plays a role in the interaction of the subunits to form higher oligomeric structures. The N-terminal catalytic domain is more stable and less prone to aggregate than full-length enzyme and is thus potentially more suitable for structure determination by X-ray crystallography. Comparisons of the yeast and human enzymes reveal significant differences in catalytic and regulatory properties.