title: Allosteric communication between the pyridoxal 5'-phosphate (PLP) and heme sites in the H2S generator human cystathionine β-synthase.
abstract: Human cystathionine β-synthase (CBS) is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ~2- to ~500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H(2)S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H(2)S-generating reactions catalyzed by CBS.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0600, USA.
authors.forename: Pramod Kumar
authors.initials: PK
authors.lastname: Yadav
authors.forename: Peter
authors.initials: P
authors.lastname: Xie
authors.forename: Ruma
authors.initials: R
authors.lastname: Banerjee
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites / Q000235:genetics; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006712:Homocystinuria / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D050050:Protein Carbonylation; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D013912:Threonine; D006418:Heme; D011732:Pyridoxal Phosphate; D012436:S-Adenosylmethionine; D006861:Hydrogen Peroxide; D007501:Iron; D003541:Cystathionine beta-Synthase