Vitamin B 12 Mechanism. Wan-Chun Chung 4/18/2011

Transcription

1 Vitamin B 12 Mechanism Wan-Chun Chung 4/18/2011

2 utline Introduction Isomerase mechanism problems Methyltranferase mechanism problems Dehalogenase mechanism 2

3 Introduction Brain, nervous system, blood formation, metabolism, DA synthesis Antipernicious anemia factor First water stable organometallic complex Cofactor of 1.adenosylcobalamin-dependent isomerases 2.methylcobalamin-dependent methyltransferases 3.dehalogenases oloenzyme : apoenzyme+cofactor 3

4 2 C 2 C 2 C 3 C 3 C P C 3 C 3 C 3 R Co C 3 C 2 C 3 C 3 C 3 C 2 C 3 C 3 C 2 nucleotide Structure Corrin ring R= deoxyadenosyl C -, -, 2 C 2 Dimethylbenzimidazole Base-on/base-off 2 cobalamin 4

5 Three main types Isomerase X C C Z X C C Z Diol dehydrase, ammonia lyase, glutamate mutase, methylmalonyl CoA mutase, ribonucleotide reductase Methyltransferase R C 3 R' R R' C 3 Methionine synthase, acetyl-coa synthase Reductive dehalogenase R Cl 2e R Cl anaerobic dehalogenase Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72,

6 Isomerase X C C Z X C C Z Class I : Base-off/is-on Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72,

7 omolysis v.s. eterolysis Co III C 2 Ado Co II C 2 Ado R Co II R C 3 Ado C Co C Co C Co B B B Co III C 2 Ado Co I C 2 Ado R Co I R C 3 Ado Co III d 6, l.s. 18 e - Co II d 7, l.s. 17 e - Co I d 8, l.s. 16 e - Co II EPR evidence ( also substrate radical) Some reactions have inversion while some others have retention on substrate no planar intermediate Diol dehydrase : ()CC(=) very stable, deactivates cycle omolytic pathway! Jordan, R. B. Reaction Mechanisms of Inorganic and rganometallic Systems; xford,

8 Important role of protein: constrains and protects reactive intermediate (. R), inhibits unwanted side reactions Base-off/his-on o. C 2 Ado observed it s reactive and shortlived k>300 s -1 stopped-flow KIE=30 not only Co-C cleavage but coupled with hydrogen abstraction is RDS Jordan, R. B. Reaction Mechanisms of Inorganic and rganometallic Systems; xford,

9 Isomerase Class II : Base-on Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72,

10 Problems Thermal stability of Co-C bond compare to high reactivity of enzymic reaction 1. Co (D) 2 Co (or (DMG) 2 Co) 2. L(DMG) 2 Co-C 3 stretching frequency, L affects the stability of Co II alpern, J.; g, F.T.T; Rempe, G. L. J Am Chem Soc,1979, 105, 7124 Jordan, R. B. Reaction Mechanisms of Inorganic and rganometallic Systems; xford,,

11 In water heterolysis takes place Finke & ay found Δ * =33±2 kcal/mol ΔS * = 11±3 cal/mol*k k=1*10-9 s -1 (cp. k>300 s -1 ) enzyme distorts the coenzyme so that the homolysis is 15 kcal/mol more favored ay, B. P.; Finke.R. G. J. Am. Chem. Soc. 1986, 108,

12 Problems Limited precedent for radical rearrangement Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72,

13 Methyltransferase Methionine synthase 2 C 3 R S 3 - C homocysteine 3-4 folate 4 folate methionine 2 R 3 CS 3-2 C 3 R Co I - S C R C 3 Co III B - - S 3 Shriver textbook 13

14 ucleophile attack vs xidative addition For overall reaction, the methyl configuration is retention both steps retention or inversion Absence of EPR signal for Co II or organic radical no homolytic Co-C cleavage xidative addition needs two cis vacant site impossible Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72,

15 Problems Methyl donor (Me- 4 folate) is too electron rich to undergo u attack protonation C 3 R 2 15

16 Problems Methyl donor (Me- 4 folate) is too electron rich to undergo u attack protonation Methyl acceptor (thiol of homocysteine, a u) needs to be deprotonated, pk a =10 Zinc help EXAFS evidence of Zn(II)S 3 add homocysteine Zn(II)S 4 16

17 Modified mechanism + Me- 4 folate C 3 Co Co Co 3 C Zn II (S) 3 C 3 Co SR - + Zn II (S) 3 S R C 3 Co Zn II (S) 3 3 C S R Co 17

18 Acetogenesis Wood-Ljungdahl Pathway ACS: acetyl-coa synthase CFeSP : corrinoid iron-sulfur protein contains 4Fe-4S cluster -Demethylase: couples the demethylation of an aromatic methyl ether to the formation of C 3 -folate CD : C dehydrogenase S P P P 2 Acetyl-CoA Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72,

19 Methanogenesis SCoA C 4 Reverse of acetyl-coa synthesis Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72,

20 Reductive Dehalogenase R Cl 2e R Cl Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem 2003, 72,

21 Birch reduction Cl R Cl R + Cl R Cl R R e - e - Cl - Path A -organocobalt complex formation or path B -just an electron donor? o intermediate has been found Low potential reductants drive the reaction Co I is the active species [Co I ] disappear as [Co II ] appear path B k cat ( 2 ) = 2.3* k cat (D 2 ) 21

22 Isotope k cat ( 2 ) /k cat (D 2 ) = 2.3 Proton inventory is linear D from solvent is incorporated in the organics Single proton is transferred in a partially rate limiting reaction Isotope inventory plot for the dehalogenation of 3-chloro-4-hydroxybenzoate deprotonation of an active-site water Krasotkina, J.; Walters, T.; Maruya, K. A.; Ragsdale, S. W. J. Biol. Chem. 2001, 276,