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.     

The heme of cystathionine beta-synthase likely undergoes a thermally induced redox-mediated ligand switch

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-dependent enzyme that catalyzes the condensation of homocysteine and serine to form cystathionine. Human CBS is unique in that heme is also required for maximal activity, although the function of heme in this enzyme is presently unclear. The study presented herein reveals that the heme of human CBS undergoes a coordination change upon reduction at elevated temperatures. We have termed this new species "CBS424" and demonstrate that its formation is likely irreversible when pH 9 Fe(III) CBS is reduced at moderately elevated temperatures (approximately 40 degrees C and higher) or when pH 9 Fe(II) CBS is heated to similar temperatures. Spectroscopic techniques, including resonance Raman, electronic absorption, and variable temperature/variable field magnetic circular dichroism spectroscopy, provide strong evidence that CBS424 is coordinated by two neutral donor ligands. It appears likely that the native cysteine(thiolate) heme ligand is displaced by an endogenous neutral donor upon conversion to CBS424. This behavior is consistent with other six-coordinate, cysteine(thiolate)-ligated heme centers, which seek to avoid this coordination structure in the Fe(II) state. Functional assays show that CBS424 is inactive and suggest that the ligand switch is responsible for eliminating enzyme activity. When this investigation is taken together with other functional studies of CBS, it provides strong evidence that coordination of Cys52 to the heme iron is crucial for full activity in this enzyme. We hypothesize that cysteine displacement may serve as a mechanism for CBS inactivation and that second-sphere interactions of the Cys52 thiolate with surrounding residues are responsible for communicating the heme ligand displacement to the CBS active site.     

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.     

Solvent-accessible cysteines in human cystathionine beta-synthase: crucial role of cysteine 431 in S-adenosyl-L-methionine binding

Cystathionine beta-synthase (CBS) is a tetrameric heme protein that catalyzes the PLP-dependent condensation of serine and homocysteine to cystathionine. CBS occupies a crucial regulatory position between the methionine cycle and transsulfuration. Human CBS contains 11 cysteine residues that are highly conserved in mammals but completely absent in the yeast enzyme, which catalyzes an identical reaction, suggesting a possible regulatory role for some of these residues. In this report, we demonstrate that in both the presence and absence of the CBS allosteric regulator S-adenosyl-l-methionine (AdoMet), only C15 and C431 of human CBS are solvent accessible. Mutagenesis of C15 to serine did not affect catalysis or AdoMet activation but significantly reduced aggregation of the purified enzyme in vitro. Mutagenesis of C431 resulted in a constitutively activated form of CBS that could not be further activated by either AdoMet or thermal activation. We and others have previously reported a number of C-terminal CBS point mutations that result in a decreased or abolished response to AdoMet. In contrast to all of these previously investigated CBS mutants, the C431 mutant form of CBS was unable to bind AdoMet, indicating that either this residue is directly involved in AdoMet binding or its absence induces a conformational change that destroys the integrity of the binding site for this regulatory ligand.     

Active cystathionine beta-synthase can be expressed in heme-free systems in the presence of metal-substituted porphyrins or a chemical chaperone

Cystathionine beta-synthase (CBS), a key enzyme in the metabolism of homocysteine, has previously been shown to require a heme co-factor for maximal activity. However, the biochemical function of the CBS heme is not well defined. Here, we show that expression of human CBS in heme-deficient strains of Saccharomyces cerevisiae and Escherichia coli results in production of an enzyme that is misfolded and degraded. Addition of exogenous heme, porphyrins with non-iron metal, or porphyrin lacking metal entirely produced stable and active CBS enzyme. Purification of recombinant CBS enzyme expressed in the presence of various metalloporphyrins confirmed that Mn(III) and Co(III) had 30-60% of the specific activity of Fe(III)-CBS, and still responded to allosteric activation by S-adenosyl-L-methionine. Treatment of S. cerevisiae with the chemical chaperone trimethylamine-N-oxide resulted in near complete restoration of function to human CBS produced in a heme-deficient strain. Taken together, these results suggest that porphyrin moiety of the heme plays a critical role in proper CBS folding and assembly, but that the metal ion is not essential for this function or for allosteric regulation by S-adenosyl-L-methionine.     

Transsulfuration in Saccharomyces cerevisiae is not dependent on heme: purification and characterization of recombinant yeast cystathionine beta-synthase

Cystathionine beta-synthase [CBS; L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration in both yeast and humans. It has been established previously that human CBS is a hemeprotein but although the heme group appears to be essential for CBS activity, the exact function of the heme group is unknown. CBS activity is absent in heme deficient strains of Saccharomyces cerevisiae grown without heme supplementation. CBS activity can be restored by supplementing these strains with heme, implying that there is a heme requirement for yeast CBS. We subcloned, overexpressed and purified yeast CBS. The yeast enzyme shows absolute pyridoxal 5'-phosphate (PLP) dependence for activity but we could find no evidence for the presence of a heme group. Given the degree of sequence and mechanistic similarity between yeast and human CBS, this result indicates that heme is unlikely to play a direct catalytic role in the human CBS reaction mechanism. Further characterization revealed that, in contrast to human CBS, S-adenosylmethionine (AdoMet) does not activate yeast CBS. Yeast CBS was found to be coordinately regulated with proliferation in S. cerevisiae. This finding is the most likely explanation of the observed apparent heme dependence of transsulfuration in vivo.     

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.     

Modulation of Cystathionine β-Synthase Activity by the Arg-51 and Arg-224 Mutations

Human cystathionine β-synthase (CBS) catalyzes a pyridoxal 5′-phosphate (PLP) dependent β-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 Å away from heme. A possible explanation of our results is discussed on the basis of CBS structure.     

Reversible heme-dependent regulation of human cystathionine β-synthase by a flavoprotein oxidoreductase

Human CBS is a PLP-dependent enzyme that clears homocysteine, gates the flow of sulfur into glutathione, and contributes to the biogenesis of H(2)S. The presence of a heme cofactor in CBS is enigmatic, and its conversion from the ferric- to ferrous-CO state inhibits enzyme activity. The low heme redox potential (-350 mV) has raised questions about the feasibility of the ferrous-CO state forming under physiological conditions. Herein, we provide the first evidence of reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH. These data provide a mechanism for cross talk between two gas-signaling systems, CO and H(2)S, via heme-mediated allosteric regulation of CBS.     

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.     

Dynamics of carbon monoxide binding to cystathionine beta-synthase

Cystathionine beta-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 microm(-1). Stopped flow measurements reveal slow CO association (0.0166 s(-1)) limited by dissociation of the endogenous ligand, Cys-52. Rebinding of CO and of Cys-52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys-52 rebinding rate, 4000 s(-1), is essentially unchanged between pH 7.6 and 10.5, indicating that the pK(a) of Cys-52 is shifted below pH 7.6. This effect is attributed to the nearby Arg-266 residue, which is proposed to form a salt bridge with the dissociated Cys-52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg-266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.     

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.     

Purification and characterization of cystathionine β-synthase bearing a cobalt protoporphyrin

Human cystathionine β-synthase (CBS), a pivotal enzyme in the metabolism of homocysteine, is a pyridoxal-5′-phosphate-dependent enzyme that also contains heme, a second cofactor whose function is still unclear. One strategy for elucidation of heme function is its replacement with different metalloporphyrins or with porphyrins containing different substituent groups. This paper describes a novel expression approach and purification of cobalt CBS (CoCBS), which results in a high yield of fully active, high purity enzyme, in which heme is substituted by Co-protoporphyrin IX (CoPPIX). Metal content analysis showed that the enzyme contained 92% cobalt and 8% iron. CoCBS was indistinguishable from wild-type FeCBS in its activity, tetrameric oligomerization, PLP saturation and responsiveness to the allosteric activator, S-adenosyl-l-methionine. The observed biochemical and spectral characteristics of CoCBS provide further support for the suggestion that heme is involved in structural integrity and folding of this unusual enzyme.     

Effect of the Disease-Causing R266K Mutation on the Heme and PLP Environments of Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme b cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme. Using rR spectroscopy, we show that the Fe(III)-Cys(thiolate) bond is longer and weaker in R266K, as evidenced by an 8 cm(-1) downshift in the ν(Fe-S) resonance. Presence of this longer and weaker Fe(III)-Cys(thiolate) bond is correlated with alteration of the fluorescence spectrum of the active PLP ketoenamine tautomer. Activity data demonstrate that, relative to WT, the R266K variant is more impaired in the alternative cysteine-synthesis reaction than in the canonical cystathionine-synthesis reaction. This diminished cysteine synthesis activity and a greater sensitivity to exogenous PLP correlate with the change in PLP environment. Fe-S(Cys) bond weakening causes a nearly 300-fold increase in the rate of ligand switching upon reduction of the R266K heme. Combined, these data demonstrate cross talk between the heme and PLP active sites, consistent with previous proposals, revealing that alteration of the Arg(266)-Cys(52) interaction affects PLP-dependent activity and dramatically destabilizes the ferrous thiolate-ligated heme complex, underscoring the importance of this hydrogen-bonding residue pair.     

Folding and activity of mutant cystathionine β-synthase depends on the position and nature of the purification tag: characterization of the R266K CBS mutant

Cystathionine β-synthase (CBS), a heme-containing pyridoxal-5-phosphate (PLP)-dependent enzyme, catalyzes the condensation of serine and homocysteine to yield cystathionine. Missense mutations in CBS, the most common cause of homocystinuria, often result in misfolded proteins. Arginine 266, where the pathogenic missense mutation R266K was identified, appears to be involved in the communication between heme and the PLP-containing catalytic center. Here, we assessed the effect of a short affinity tag (6xHis) compared to a bulky fusion partner (glutathione S-transferase - GST) on CBS wild type (WT) and R266K mutant enzyme properties. While WT CBS was successfully expressed either in conjunction with a GST or with a 6xHis tag, the mutant R266K CBS had no activity, did not form native tetramers and did not respond to chemical chaperone treatment when expressed with a GST fusion partner. Interestingly, expression of R266K CBS constructs with a 6xHis tag at either end yielded active enzymes. The purified, predominantly tetrameric, R266K CBS with a C-terminal 6xHis tag had ～82% of the activity of a corresponding WT CBS construct. Results from thermal pre-treatment of the enzyme and the denaturation profile of R266K suggests a lower thermal stability of the mutant enzyme compared to WT, presumably due to a disturbed heme environment.     

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.     

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.     

S-Adenosyl-l-methionine Modulates CO and NO? Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is a key enzyme in human (patho)physiology with a central role in hydrogen sulfide metabolism. The enzyme is composed of a pyridoxal 5′-phosphate-binding catalytic domain, flanked by the following two domains: a heme-binding N-terminal domain and a regulatory C-terminal domain binding S-adenosyl-l-methionine (AdoMet). CO or NO^• binding at the ferrous heme negatively modulates the enzyme activity. Conversely, AdoMet binding stimulates CBS activity. Here, we provide experimental evidence for a functional communication between the two domains. We report that AdoMet binding significantly enhances CBS inhibition by CO. Consistently, we observed increased affinity (∼5-fold) and faster association (∼10-fold) of CO to the ferrous heme at physiological AdoMet concentrations. NO^• binding to reduced CBS was also enhanced by AdoMet, although to a lesser extent (∼2-fold higher affinity) as compared with CO. Importantly, CO and NO^• binding was unchanged by AdoMet in a truncated form of CBS lacking the C-terminal regulatory domain. These unprecedented observations demonstrate that CBS activation by AdoMet puzzlingly sensitizes the enzyme toward inhibition by exogenous ligands, like CO and NO^•. This further supports the notion that CBS regulation is a complex process, involving the concerted action of multiple physiologically relevant effectors.     

Cross-talk between the catalytic core and the regulatory domain in cystathionine β-synthase: study by differential covalent labeling and computational modeling

Cystathionine β-synthase (CBS) is a modular enzyme which catalyzes condensation of serine with homocysteine. Cross-talk between the catalytic core and the C-terminal regulatory domain modulates the enzyme activity. The regulatory domain imposes an autoinhibition action that is alleviated by S-adenosyl-l-methionine (AdoMet) binding, by deletion of the C-terminal regulatory module, or by thermal activation. The atomic mechanisms of the CBS allostery have not yet been sufficiently explained. Using pulse proteolysis in urea gradient and proteolytic kinetics with thermolysin under native conditions, we demonstrated that autoinhibition is associated with changes in conformational stability and with sterical hindrance of the catalytic core. To determine the contact area between the catalytic core and the autoinhibitory module of the CBS protein, we compared side-chain reactivity of the truncated CBS lacking the regulatory domain (45CBS) and of the full-length enzyme (wtCBS) using covalent labeling by six different modification agents and subsequent mass spectrometry. Fifty modification sites were identified in 45CBS, and four of them were not labeled in wtCBS. One differentially reactive site (cluster W408/W409/W410) is a part of the linker between the domains. The other three residues (K172 and/or K177, R336, and K384) are located in the same region of the 45CBS crystal structure; computational modeling showed that these amino acid side chains potentially form a regulatory interface in CBS protein. Subtle differences at CBS surface indicate that enzyme activity is not regulated by conformational conversions but more likely by different allosteric mechanisms.     

Purification and characterization of the wild type and truncated human cystathionine beta-synthase enzymes expressed in E. coli

In this paper, we describe the expression and characterization of recombinant human cystathionine β-synthase (CBS) in Escherichia coli. We have used a glutathione-S-transferase (GST) fusion protein vector and incorporated a cleavage site with a long hinge region which allows for the independent folding of CBS and its fusion partner. In addition, our construct has the added benefit of yielding a purified CBS which only contains one extra glycine amino acid residue at the N-terminus. In our two-step purification procedure we are able to obtain a highly pure enzyme in sufficient quantities for crystallography and other physical chemical methods. We have investigated the biochemical and catalytic properties of purified full-length human CBS and of two truncation mutants lacking the C-terminal domain or both the N-terminal heme-binding and the C-terminal regulatory regions. Specifically, we have determined the pH optima of the different CBS forms and their kinetic and spectral properties. The full-length and the C-terminally truncated enzyme had a broad pH 8.5 optimum while the pH optimum of the N- and C- terminally truncated enzyme was sharp and shifted to pH 9. Furthermore, we have shown unequivocally that CBS binds one mole of heme per subunit by determining both the heme and the iron content of the enzyme. The activity of the enzyme was unaffected by the redox status of the heme iron. Finally, we show that CBS is stimulated by S-adenosyl- l-methionine but not its analogs.