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).
Taoka
Shinichi
S
Biochemistry Department, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Banerjee
Ruma
R
D002384:Catalysis; D003540:Cystathionine / Q000737:chemistry; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006418:Heme / Q000737:chemistry; D006710:Homocysteine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry; D013997:Time Factors
D006710:Homocysteine; D003540:Cystathionine; D006418:Heme; D012694:Serine; D003541:Cystathionine beta-Synthase