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

Cofactors, concluded

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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 - PowerPoint PPT Presentation

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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