Cross-talk between the catalytic core and the regulatory domain in cystathionine β-synthase: study by differential covalent labeling and computational modeling.
Cystathionine β-synthase (CBS) is a modular enzyme which catalyzes condensation of serine with homocysteine. Cross-talk between the catalytic core and the C-terminal regulatory domain modulates the enzyme activity. The regulatory domain imposes an autoinhibition action that is alleviated by S-adenosyl-l-methionine (AdoMet) binding, by deletion of the C-terminal regulatory module, or by thermal activation. The atomic mechanisms of the CBS allostery have not yet been sufficiently explained. Using pulse proteolysis in urea gradient and proteolytic kinetics with thermolysin under native conditions, we demonstrated that autoinhibition is associated with changes in conformational stability and with sterical hindrance of the catalytic core. To determine the contact area between the catalytic core and the autoinhibitory module of the CBS protein, we compared side-chain reactivity of the truncated CBS lacking the regulatory domain (45CBS) and of the full-length enzyme (wtCBS) using covalent labeling by six different modification agents and subsequent mass spectrometry. Fifty modification sites were identified in 45CBS, and four of them were not labeled in wtCBS. One differentially reactive site (cluster W408/W409/W410) is a part of the linker between the domains. The other three residues (K172 and/or K177, R336, and K384) are located in the same region of the 45CBS crystal structure; computational modeling showed that these amino acid side chains potentially form a regulatory interface in CBS protein. Subtle differences at CBS surface indicate that enzyme activity is not regulated by conformational conversions but more likely by different allosteric mechanisms.
Institute of Inherited Metabolic Disorders, First Medical Faculty, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, Prague 2, 128 00 Czech Republic.
Ales
A
Hnízda
Vojtech
V
Spiwok
Vojtech
V
Jurga
Viktor
V
Kozich
Milan
M
Kodícek
Jan P
JP
Kraus
D000495:Allosteric Site; D020134:Catalytic Domain / Q000502:physiology*; D019295:Computational Biology / Q000379:methods*; D018360:Crystallography, X-Ray; D003541:Cystathionine beta-Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000502:physiology; D020239:Receptor Cross-Talk / Q000502:physiology*
D003541:Cystathionine beta-Synthase