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11/30/2010Biochemistry: Metabolism IV
Cofactors, concluded
Andy HowardIntroductory Biochemistry
30 November 2010
11/30/2010Biochemistry: Metabolism IV
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Metabolism depends strongly on cofactors We’ll attend to the reality that a lot of the versatility of enzymes depends on their incorporation of cofactors
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Cofactor topics Cosubstrates
ATP and relatives
Redox cosubstrates
Prosthetic groups Thioesters Redox prosthetic groups
Prosthetic Groups, concluded TPP PLP Other prosthetic groups
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Major cosubstrates Facilitate group transfers, mostly small groups
Oxidation-reduction participantsCosubstrate Source FunctionATP Transfer P,NucleotideS-adenosylMet Methyl transferUDP-glucose Glycosyl transferNAD,NADP Niacin 2-electron redoxCoenzyme A Pantothenate Acyl transferTetrahydrofolate Folate 1Carbon
transferUbiquinone Lipid-soluble e- carrier
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Major prosthetic groups Transfer of larger groups
One- or two-electron redox changesProsth.gp. Source FunctionFMN, FAD Riboflavin1e- and 2e- redox transfersTPP Thiamine 2-Carbon transfers with C=OPLP PyridoxineAmino acid group transfersBiotin Biotin Carboxylation, COO- transferAdenosyl- Cobalamin Intramolec. rearrangements cobalaminMeCobal. Cobalamin Methyl-group transfersLipoamide Transfer from TPPRetinal Vitamin A VisionVitamin K Vitamin K Carboxylation of glu
residues
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NAD+ and NADP+
Net charge isn’t really >0 ;the + is just a reminder that the nicotinamide ring is positively charged
Most important cosubstrates in oxidation-reduction reactions in aerobic organisms
Structure courtesy of Sergio Marchesini, U. Brescia
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Differences between them
The chemical difference is in the phosphorylation of the 2’ phosphate group of the ribose moiety
The functional difference is that NAD+ is usually associated with catabolic reactions and NADP+ is usually associated with anabolic reactions
Therefore often NAD+ and NADPH are reactants and NADH and NADP+ are products
Exceptions: photosynthesis and ETC!
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How do we get back to the starting point? NADH is often oxidized back to NAD+ as part of the electron-transport chain
NADPH is created via photosynthesis Imbalances can be addressed viaNAD Kinase (S.Kawai et al (2005), J.Biol.Chem. 280:39200) and NADP phosphatase
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Reduced forms of NAD(P)
Reduction occurs on the nicotinamide ring
Ring is no longer net-positive
Ring is still planar but the two hydrogens on the para carbon are not
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NADPH Provides reducing power for anabolic reactions
Often converting highly oxidized sugar precursors into less oxidized molecules
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FAD and FMN Flavin group based on riboflavin Alternate participants in redox reactions Prosthetic groups
tightly but noncovalently bound to their enzymes That protects against wasteful reoxidation of reduced forms
FADH2 is weaker reducing agent than NADH:when used as an energy source, it yields 1.5 ATP per oxidation, whereas NADH yields 2.5
These are capable of one-electron oxidations and reductions
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FAD and FMN structures FAD has an AMP attached P to P
Structure courtesyPaisley University
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Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University
FMN/FAD redox forms
Two-electron version: H+ + :H- transferred
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iClicker quiz question 1
Based on what you have learned, would you expect glycogen synthase to be activated or inhibited by phosphorylation?
(a) activated (b) inhibited (c) neither (d) insufficient information to tell
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iClicker quiz question 2
What would you expect to be the phosphate donor in the NAD kinase reaction?
(a) free phosphate (b) pyrophosphate (c) ATP (d) pyridoxal phosphate
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Thiamine Pyrophosphate
Based on thiamine, vitamin B1 Carboxylases and oxidative decarboxylases use this coenzyme
So do transketolases (move 2 carbons at a time between sugars with keto groups)
Thiazolium ring is reactive center:pKa drops from 15 in H2O to 6 in enzyme
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TPP Derived as in fig.17.17
We already talked about decarboxylations of -ketoacids, e.g.pyruvate + H+ acetaldehyde + CO2
Formation and cleavage of -hydroxylactones &-hydroxyacids:2 pyruvate + H+ acetolactate + CO2
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TPP reactions
Diagram courtesy ofOklahoma State U.Biochemistry program
pyrimidine
thiazolium
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Pyridoxal phosphate
PLP is prosthetic group for many amino-acid-related enzymes, particularly transaminations
That’s how a lot of -amino acids are synthesized from the corresponding -ketoacids:H3N+—CHR1—COO- + O=CHR2-COO- O=CHR1-COO- + H3N+—CHR2—COO-
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How PLP functions
Carbonyl group of PLP bound as a Schiff base (imine) to -amino group of lysine at active site
First step is always formation of external aldimine; goes through gem-diamine intermediate to internal aldimine
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PLP Remember we said it gets used in a lot of transaminations
We should consider its chemistry and its other roles in pathways
To start with: it exists in 2 tautomeric forms
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PLP:Non-transamination reactions -decarboxylation:
-amino acid + H+ CO2 + H3N+-CH2-R
-decarboxylation Others listed in fig. 17.26
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PLP intermediates
See fig.17.27: it’s complex but important
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Biotin Rarity: vitamin is the prosthetic group
Used in reactions that transfer carboxyl groups
… and in ATP-dependent carboxylations
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Biotin reactivity Covalently bound to active-site
lysines to form species called biocytin
Pyruvate carboxylase is characteristic reaction:
Diagram courtesyUniversity of Virginia Biochemistry
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Tetrahydrofolate Primary donor of one-carbon units(formyl, methylene, methyl)
Supplies methyl group for thymidylate
Dihydrofolate reductase (DHFR) is an interesting drug target Methotrexate as cancer chemotherapeutic: cancer needs more thymidylate than healthy cells
Trimethoprim as antibacterial:Bacterial DHFR is somewhat different from eucaryotic DHFR because bacteria derive DHF from other sources; humans get it from folate
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THF structure and function
Figure courtesy horticulture program, Purdue
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Tetrahydrofolate variations
-2 oxidation state:methyl donor from N5-methyl-THF
0 oxidation state: methylene donor from N5,N10-methylene-THF
+2 oxidation state: formyl (-CH=O) from N5-formyl-THF and N10-formyl-THF
Formimino (-CH=NH) from N5-formimino-THF
Methenyl (-CH=) from N5,N10-methenyl-THF
See table 17.6 for specifics
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Thymidylate cycle!
Remember that thymidine isthe rate-limiting reagent in DNA synthesis
Thymidylate derived from uridylate in a 5,10-methylenetetrahydrofolate dependent reaction:uridylate + 5,10-meTHF thymidylate + dihydrofolate
Catalyzed by thymidylate synthase Rest of cycle gets DHF reconverted into 5,10-meTHF
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The restorative reactions
Dihydrofolate reductase (DHFR): DHF + NADH THF + NAD Enzyme is popular drug target, as suggested
Serine hydroxymethyltransferase (SHMT): THF + serine 5,10meTHF + glycine This also serves as a common synthetic pathway for creating glycine from serine
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Cobalamin Largest B vitamin Structure related to heme but missing one carbon in ring structure
Cobalt bound in core of ring system
Involved in enzymatic rearrangements Catabolism of odd-chain fatty acids Methylation of homocysteine Reductive dehalogenation
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Adenosyl-Cobalamin
Diagram courtesy of Swiss Food News
“Missing” carbon
ReactiveCo-C bond
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Lipoamide Protein-bound form of lipoic acid Contains five-membered disulfide ring Covalently bound via amide to protein lysine sidechain
Involved in swinging arm between active sites in multienzyme complexes
Disulfide breaks, re-forms during activity
Examples: pyruvate dehydrogenase complex, -ketoglutarate dehydrogenase
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Lipoamide 2e- reduction thioester starting point
Fig. Courtesy Biochem and Biophysics program, Rensselaer
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iClicker quiz question 3
Which coenzyme would you expect would be required for the reactionoxaloacetate + glutamate aspartate + -ketoglutarate?(a) ascorbate(b) PLP(c) thiamine pyrophosphate(d) NAD(e) none of the above
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iClicker question 4
A transamination is (a) A simple substitution of N for O
(b) A redox reaction (c) Possible only at high pH (d) Energetically unfavorable (e) none of the above