11/30/2010Biochemistry: Metabolism IV Cofactors, concluded Andy Howard Introductory Biochemistry 30 November 2010

11/30/2010Biochemistry: Metabolism IV Page 2 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 3 of 36 Cofactor topics Cosubstrates ATP and relatives Redox cosubstrates Prosthetic groups Thioesters Redox prosthetic groups Prosthetic Groups, concluded TPP PLP Other prosthetic groups

11/30/2010Biochemistry: Metabolism IV Page 4 of 36 Major cosubstrates Facilitate group transfers, mostly small groups Oxidation-reduction participants CosubstrateSourceFunction ATPTransfer P,Nucleotide S-adenosylMetMethyl transfer UDP-glucoseGlycosyl transfer NAD,NADPNiacin2-electron redox Coenzyme APantothenateAcyl transfer TetrahydrofolateFolate1Carbon transfer UbiquinoneLipid-soluble e - carrier

11/30/2010Biochemistry: Metabolism IV Page 5 of 36 Major prosthetic groups Transfer of larger groups One- or two-electron redox changes Prosth.gp.SourceFunction FMN, FADRiboflavin1e - and 2e - redox transfers TPPThiamine2-Carbon transfers with C=O PLPPyridoxineAmino acid group transfers BiotinBiotinCarboxylation, COO - transfer Adenosyl-CobalaminIntramolec. rearrangements cobalamin MeCobal.CobalaminMethyl-group transfers LipoamideTransfer from TPP RetinalVitamin AVision Vitamin KVitamin KCarboxylation of glu residues

11/30/2010Biochemistry: Metabolism IV Page 6 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 7 of 36 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!

11/30/2010Biochemistry: Metabolism IV Page 8 of 36 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 via NAD Kinase (S.Kawai et al (2005), J.Biol.Chem. 280:39200) and NADP phosphatase

11/30/2010Biochemistry: Metabolism IV Page 9 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 10 of 36 NADPH Provides reducing power for anabolic reactions Often converting highly oxidized sugar precursors into less oxidized molecules

11/30/2010Biochemistry: Metabolism IV Page 11 of 36 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 FADH 2 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

11/30/2010Biochemistry: Metabolism IV Page 12 of 36 FAD and FMN structures FAD has an AMP attached P to P Structure courtesy Paisley University

11/30/2010Biochemistry: Metabolism IV Page 13 of 36 Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University FMN/FAD redox forms Two-electron version: H + + :H - transferred

11/30/2010Biochemistry: Metabolism IV Page 14 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 15 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 16 of 36 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: pK a drops from 15 in H 2 O to 6 in enzyme

11/30/2010Biochemistry: Metabolism IV Page 17 of 36 TPP Derived as in fig We already talked about decarboxylations of  - ketoacids, e.g. pyruvate + H +  acetaldehyde + CO 2 Formation and cleavage of  -hydroxylactones &  -hydroxyacids: 2 pyruvate + H +  acetolactate + CO 2

11/30/2010Biochemistry: Metabolism IV Page 18 of 36 TPP reactions Diagram courtesy of Oklahoma State U. Biochemistry program pyrimidine thiazolium

11/30/2010Biochemistry: Metabolism IV Page 19 of 36 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: H 3 N + —CHR 1 —COO - + O=CHR 2 -COO -  O=CHR 1 -COO - + H 3 N + —CHR 2 —COO -

11/30/2010Biochemistry: Metabolism IV Page 20 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 21 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 22 of 36 PLP: Non-transamination reactions  -decarboxylation:  -amino acid + H +  CO 2 + H 3 N + -CH 2 -R  -decarboxylation Others listed in fig

11/30/2010Biochemistry: Metabolism IV Page 23 of 36 PLP intermediates See fig.17.27: it’s complex but important

11/30/2010Biochemistry: Metabolism IV Page 24 of 36 Biotin Rarity: vitamin is the prosthetic group Used in reactions that transfer carboxyl groups … and in ATP-dependent carboxylations

11/30/2010Biochemistry: Metabolism IV Page 25 of 36 Biotin reactivity Covalently bound to active-site lysines to form species called biocytin Pyruvate carboxylase is characteristic reaction: Diagram courtesy University of Virginia Biochemistry

11/30/2010Biochemistry: Metabolism IV Page 26 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 27 of 36 THF structure and function Figure courtesy horticulture program, Purdue

11/30/2010Biochemistry: Metabolism IV Page 28 of 36 Tetrahydrofolate variations -2 oxidation state: methyl donor from N 5 -methyl-THF 0 oxidation state: methylene donor from N 5,N 10 -methylene-THF +2 oxidation state: formyl (-CH=O) from N 5 -formyl-THF and N 10 - formyl-THF Formimino (-CH=NH) from N5-formimino-THF Methenyl (-CH=) from N5,N10-methenyl-THF See table 17.6 for specifics

11/30/2010Biochemistry: Metabolism IV Page 29 of 36 Thymidylate cycle! Remember that thymidine is the 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

11/30/2010Biochemistry: Metabolism IV Page 30 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 31 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 32 of 36 Adenosyl- Cobalamin Diagram courtesy of Swiss Food News “Missing” carbon Reactive Co-C bond

11/30/2010Biochemistry: Metabolism IV Page 33 of 36 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

11/30/2010Biochemistry: Metabolism IV Page 34 of 36 Lipoamide 2e - reduction thioester starting point Fig. Courtesy Biochem and Biophysics program, Rensselaer

11/30/2010Biochemistry: Metabolism IV Page 35 of 36 iClicker quiz question 3 Which coenzyme would you expect would be required for the reaction oxaloacetate + glutamate  aspartate +  -ketoglutarate? (a) ascorbate (b) PLP (c) thiamine pyrophosphate (d) NAD (e) none of the above

11/30/2010Biochemistry: Metabolism IV Page 36 of 36 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