Inhibierung des humanen B₁₂‐verarbeitenden Enzyms CblC durch Antivitamine B₁₂ – Kristallstruktur des inaktiven ternären Komplexes mit dem Kosubstrat Glutathion

Abstract

Antivitamine B 12 gewinnen als robuste B 12 -Dummys zunehmend biomedizinisches Interesse.D as potenzielle Antivitamin B 12 2,4-Difluorphenylethinylcobalamin (F2PhEtyCbl) wurde hergestellt, und seine 3D-Struktur in Lçsung und im Kristall wurde untersucht. Das chemisch inerte F2PhEtyCbl zeigte sich gegen Thermolyse seiner Co-C-Bindung bei 100 8 8C resistent, war stabil bei Bestrahlung mit (hellem) Tageslicht und blieb auchb ei längerer Aufbewahrung in wässriger Lçsung bei Raumtemperatur intakt. Es wurde vom humanen B 12 -verarbeitenden Enzym CblC in Gegenwart des Kosubstrats Glutathion (GSH) mit hoher Affinität( K D = 130 nm)g ebunden. F2PhEtyCbl stabilisierte den ternären Komplex von CblC mit GSH und widerstand der Verarbeitung.D ie Kristallstruktur dieses Komplexes lieferte erste Einblicke in die Wechselwirkungen eines Antivitamins B 12 mit CblC sowiei nd ie Anordnung von GSH und Base-off-Cobalamin im aktiven Zentrum dieses blockierten Enzyms.

Antivitamins B12—Some Inaugural Milestones

“…Furthermore, the fluorinated 2,4‐difluorophenyl‐derivative F2PhEtyCbl was not only light‐stable, [27] but also rather inert against acid‐induced hydrolytic cleavage of its Co−C bond, as expected [26b] . F2PhEtyCbl bound and inhibited the holoenzyme CblC loaded with the co‐substrate glutathione, allowing for a first crystal‐structure analysis of fully assembled human CblC [26b] . Investigations, not only from our laboratory, but also from the Gryko group, [28] have meanwhile expanded the methodology for the preparation of organometallic alkynyl‐cobalt‐corrinoids.…”

“…The previously unknown phenylethynyl‐cobalamin (PhEtyCbl) was prepared, which turned out to be slightly hydrolysis‐sensitive, but was light stable and thermally robust and exhibited similar binding‐affinity as CNCbl for the human proteins of B 12 ‐transport [26a] . Furthermore, the fluorinated 2,4‐difluorophenyl‐derivative F2PhEtyCbl was not only light‐stable, [27] but also rather inert against acid‐induced hydrolytic cleavage of its Co−C bond, as expected [26b] . F2PhEtyCbl bound and inhibited the holoenzyme CblC loaded with the co‐substrate glutathione, allowing for a first crystal‐structure analysis of fully assembled human CblC [26b] .…”

“…Studies with antivitamins B 12 and other structurally characterized Metbls may potentially contribute to this subject, [36] relying on two key structure‐based factors: (i) By imitating the structures of the B 12 ‐cofactors or of reactive intermediate B 12 ‐species in the course of enzyme reactions, suitably structured (inactive) B 12 ‐mimics have an excellent capacity to inhibit the corresponding enzymatic steps. Hence, for example, the Ni II ‐analogue of the cryptic intermediate Co I ‐form cob(I)alamin inhibits an AdoCbl‐generating Ado‐transferase in an in vitro study [36] (see above for corresponding pertinent findings with the alkynyl‐Cbl F2PhEtyCbl [26b] and with AdoRhbl [34] ). (ii) By mimicking the structures of the B 12 ‐type ligands in B 12 ‐dependent regulatory functions in various organisms, antivitamins B 12 are, on the other hand, presumed to simulate the availability of the corresponding physiologically active B 12 ‐derivatives, for example, via B 12 ‐riboswitches [57] and in B 12 ‐responsive regulatory proteins [51, 58] .…”