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Cofactors, concluded. Andy Howard Introductory Biochemistry 30 November 2010. 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. Cosubstrates ATP and relatives Redox cosubstrates
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Cofactors, concluded Andy HowardIntroductory Biochemistry 30 November 2010 Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
Cosubstrates ATP and relatives Redox cosubstrates Prosthetic groups Thioesters Redox prosthetic groups Prosthetic Groups, concluded TPP PLP Other prosthetic groups Cofactor topics Biochemistry: Metabolism IV
Major cosubstrates • Facilitate group transfers, mostly small groups • Oxidation-reduction participants Cosubstrate Source Function ATP Transfer P,Nucleotide S-adenosylMet Methyl transfer UDP-glucose Glycosyl transfer NAD,NADP Niacin 2-electron redox Coenzyme A Pantothenate Acyl transfer Tetrahydrofolate Folate 1Carbon transfer Ubiquinone Lipid-soluble e- carrier Biochemistry: Metabolism IV
Major prosthetic groups • Transfer of larger groups • One- or two-electron redox changes Prosth.gp. Source Function FMN, FAD Riboflavin 1e- and 2e- redox transfers TPP Thiamine 2-Carbon transfers with C=O PLP Pyridoxine Amino acid group transfers Biotin Biotin Carboxylation, COO- transfer Adenosyl- Cobalamin Intramolec. rearrangements cobalamin MeCobal. Cobalamin Methyl-group transfers Lipoamide Transfer from TPP Retinal Vitamin A Vision Vitamin K Vitamin K Carboxylation of glu residues Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
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! Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
NADPH • Provides reducing power for anabolic reactions • Often converting highly oxidized sugar precursors into less oxidized molecules Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
FAD and FMN structures • FAD has an AMP attached P to P Structure courtesyPaisley University Biochemistry: Metabolism IV
FMN/FAD redox forms • Two-electron version: H+ + :H- transferred Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
TPP reactions pyrimidine thiazolium Diagram courtesy ofOklahoma State U.Biochemistry program Biochemistry: Metabolism IV
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- Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
PLP:Non-transamination reactions • -decarboxylation:-amino acid + H+ CO2 + H3N+-CH2-R • -decarboxylation • Others listed in fig. 17.26 Biochemistry: Metabolism IV
PLP intermediates • See fig.17.27: it’s complex but important Biochemistry: Metabolism IV
Biotin • Rarity: vitamin is the prosthetic group • Used in reactions that transfer carboxyl groups • … and in ATP-dependent carboxylations Biochemistry: Metabolism IV
Biotin reactivity • Covalently bound to active-site lysines to form species called biocytin • Pyruvate carboxylase is characteristic reaction: Diagram courtesyUniversity of Virginia Biochemistry Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
THF structure and function Figure courtesy horticulture program, Purdue Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
Adenosyl-Cobalamin ReactiveCo-C bond “Missing” carbon Diagram courtesy of Swiss Food News Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV
Lipoamide 2e- reduction • thioester starting point Fig. Courtesy Biochem and Biophysics program, Rensselaer Biochemistry: Metabolism IV
iClicker quiz question 3 • Which coenzyme would you expect would be required for the reactionoxaloacetate + glutamate aspartate + a-ketoglutarate?(a) ascorbate(b) PLP(c) thiamine pyrophosphate(d) NAD(e) none of the above Biochemistry: Metabolism IV
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 Biochemistry: Metabolism IV