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.
Puranik
Mrinalini
M
Department of Chemistry, Princeton University, Princeton, New Jersey 08544.
Weeks
Colin L
CL
Department of Chemistry, Princeton University, Princeton, New Jersey 08544.
Lahaye
Dorothee
D
Department of Chemistry, Princeton University, Princeton, New Jersey 08544.
Kabil
Ömer
Ö
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664.
Taoka
Shinichi
S
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664.
Nielsen
Steen Brøndsted
SB
Department of Chemistry, Princeton University, Princeton, New Jersey 08544.
Groves
John T
JT
Department of Chemistry, Princeton University, Princeton, New Jersey 08544.
Banerjee
Ruma
R
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664. Electronic address: rbanerjee1@unl.edu.
Spiro
Thomas G
TG
Department of Chemistry, Princeton University, Princeton, New Jersey 08544. Electronic address: spiro@princeton.edu.
D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism*; D003541:Cystathionine beta-Synthase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation
D002248:Carbon Monoxide; D003541:Cystathionine beta-Synthase