Functional Properties of the Active Core of Human Cystathionine β-Synthase Crystals

Abstract

Human cystathionine ␤-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 ␣-aminoacrylate. The dissociation constant of L-serine was found to be 6 mM, close to that determined in solution. The amount of the ␣-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 ␣-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the ␣-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine ␤-synthase crystals catalyze both ␤-elimination and ␤-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine ␤-synthase.High plasmatic levels of homocysteine have recently been associated with an increased risk of cardiovascular disease (1). Homocysteine is formed from S-adenosylhomocysteine and is either removed by cystathionine ␤-synthase (EC 4.2.1.22, CBS) 1 in the trans-sulfuration pathway or remethylated to methionine in the methionine cycle. Deficiency of CBS is the major cause of inherited homocystinuria. CBS is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme catalyzing the synthesis of cystathionine from homocysteine and L-serine. The reaction proceeds via a ␤-replacement mechanism, similar to that of tryptophan synthase and O-acetylserine sulfhydrylase (2). These enzymes belong to the ␤-family and fold II type within the PLP-dependent enzymes classification (3, 4). Other members of the ␤-family are serine and threonine dehydratases. The human CBS is a 63-kDa homotetramer containing one PLP and one heme per subunit (5). Whereas the functional role of PLP is known, the role of the heme is less clear. It was demonstrated that the heme redox state affects the affinity of the enzyme for the substrates (6). Moreover, the catalytic activity of CBS is controlled by S-adenosylmethionine, which specifically binds to a C-terminal site. The trypsinolysis of a 18 kDa C-terminal fragment leads to a dimeric form, 2-fold m...

Deletion Mutagenesis of Human Cystathionine β-Synthase

“…4, B and C). Similar absorption spectra were observed for crystals of the CBS active core from which the heme cofactor had been removed by carbon monoxide (35).…”

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

“…Spectroscopic detection of the aminoacrylate species in heme-containing dCBS was accomplished by use of difference electronic absorption spectroscopy as described in Materials and Methods. Although reaction intermediates have been characterized in the non-heme CBS from yeast (18), the presence of the heme obscures the electronic spectrum of the PLP cofactor, making enzyme-monitored kinetic analyses more challenging (19,20). Conversion of intermediate 1 to the aminoacrylate species in intermediate 2 could be facilitated by activation of the βOH leaving group of substrate by Y277 and S116.…”

Rescue of Cystathionine β-Synthase (CBS) Mutants with Chemical Chaperones

“…Our previous results indicated that heme does not function in redox sensing, ligand binding, or catalysis but rather supported the structural role of heme in proper folding and/or subunit assembly (22,28,(32)(33)(34)(35). With respect to this hypothesis and the presented data, heme saturation could serve as a marker for properly folded CBS and could be indicative of the activity of purified CBS.…”