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.
Bruno
S
S
Institute of Biochemical Sciences, University of Parma, 43100 Parma, Italy.
Schiaretti
F
F
Burkhard
P
P
Kraus
J P
JP
Janosik
M
M
Mozzarelli
A
A
D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006710:Homocysteine / Q000378:metabolism; D006801:Humans; D015591:Microspectrophotometry; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D006710:Homocysteine; D006418:Heme; D012694:Serine; D011732:Pyridoxal Phosphate; D002248:Carbon Monoxide; D003541:Cystathionine beta-Synthase