11/11/2008Biochem: Cofactors,Vitamins Cofactors & Vitamins Andy Howard Introductory Biochemistry 11 November 2008

11/11/2008Biochem: Cofactors,Vitamins

Cofactors & Vitamins

Andy HowardIntroductory Biochemistry

11 November 2008

11/11/2008Biochem: Cofactors,Vitamins Page 2 of 49

What we’ll discuss Coenzymes

NAD and NADP FAD and FMN Coenzyme A TPP, PLP Biotin, THF Cobalamin Lipoamide

Vitamins Why they exist Water-soluble

vitamins Cofactor precursors Ascorbate

Fat-Soluble Vitamins


Major cosubstrates (review) Facilitate group transfers, mostly small groups Oxidation-reduction participantsCosubstrate 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


Major prosthetic groups (rvw) Transfer of larger groups One- or two-electron redox changesProsth.gp. Source FunctionFMN, FAD Riboflavin 1e- and 2e- redox transfersTPP Thiamine 2-Carbon transfers with C=OPLP Pyridoxine Amino 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


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


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


How do we get back to the starting point? NADH is often oxidized back to NAD+ as

part of the electron-transport chain Imbalances can be addressed via:

NAD Kinase (S.Kawai et al (2005), J.Biol.Chem. 280:39200)

NADP phosphatase


iClicker quiz: question 1 What would you expect to be the

phosphate donor in the NAD kinase reaction?

(a) free phosphate (b) pyrophosphate (c) ATP (d) pyridoxal phosphate


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


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 These are capable of one-electron oxidations

and reductions


FAD and FMN structures FAD has an AMP attached P to P

Structure courtesyPaisley University


Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University

FMN/FAD redox forms Two-electron version: H+ + :H- transferred


Coenzyme A Reactive portion

is free sulfhydryl at one end of the molecule

Can form thioester with acetate, etc.

Pantoate +-alanine = pantothenate

Structure courtesy ofMPB project, George Washington University

(ADP-3’P)

(Pantoate)

-alanine)

2-mercapto-ethylamine)


Thiamine Pyrophosphate Based on thiamine, vitamin B1 Many 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


TPP reactions

Diagram courtesy ofOklahoma State U.Biochemistry program

pyrimidine

thiazolium


Pyridoxal phosphate

PLP is prosthetic group for many amino-acid-related enzymes, particularly transaminations

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


Transaminations -amino acid1 +

-ketoacid2 -ketoacid1 +-amino acid2

Example: -amino acid1 = glutamate -ketoacid2 = pyruvate -ketoacid1 = a-ketoglutarate -amino acid2 = alanine

Many biosynthetic / degradative pathways for normal amino acids depend on these reactions


Biotin Rarity: vitamin is the prosthetic group Used in reactions that transfer carboxyl

groups … and in ATP-dependent carboxylations


Biotin reactivity Covalently bound to active-site lysines to

form species called biocytin Pyruvate carboxylase is characteristic

reaction:

Diagram courtesyUniversity of Virginia Biochemistry


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


THF structure and function

Figure courtesy horticulture program,

Purdue


Cobalamin Largest B vitamin Corrin ring 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


Adenosyl-Cobalamin

Diagram courtesy of Swiss Food News

“Missing” carbon

ReactiveCo-C bond


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 Disulfides break periodically Example: pyruvate dehydrogenase

complex


Lipoamide 2e- reduction Cf. Scheme 7.6: thioester starting point

Fig. Courtesy Biochem and Biophysics program, Rensselaer


iClicker revisited 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


Many cofactors are derived from vitamins We justify lumping these two topics

together because many cofactors are vitamins or are metabolites of vitamins.


Vitamins: necessary micronutrients that cannot be synthesized internally What’s a vitamin for one organism is not

for another Primates and some rodents are the only

vertebrates that don’t synthesize ascorbate

E.coli can make almost everything given energy and sources of atoms


Why wouldn’t organisms make everything?

Complex metabolites require energy for synthesis

Control of their synthesis is also metabolically expensive

Cheaper in the long run to derive these nutrients from diet


Vitamins: broad classifications Water-soluble vitamins

Coenzymes or coenzyme precursors Non-coenzymic metabolites

Fat-soluble vitamins Antioxidants Other lipidic vitamins


Are all nutrients that we can’t synthesize considered vitamins? No: If it’s required in large quantities,

it’s not a vitamin By convention, essential fatty acids like

arachidonate aren’t considered vitamins


Coenzyme precursors We’ve already outlined the fact that most

water-soluble coenzymes are derived from vitamins—typically B vitamins

Typically the dietary form can be converted by a fairly short metabolic pathway into the coenzyme form, e.g. niacin + glutamine

nicotinamide + glutamate nicotinamide + ADP-ribose NAD


The B vitamins All aqueous micronutrients Generally identified via pathologies associated

with dietary deficiencies B1: thiamin (produces TPP) B2: riboflavin (produces FAD, FMN) B3: niacin (produces NAD, NADP) B5: pantothenate (produces Coenzyme A) B6: pyridoxamine (produces PLP) B9: folate: produces THF, THF derivatives B12: cobalamin (produces adenosylcobalamin,

methylcobalamin)


Deficiency of niacin Pellagra: dermatitis, diarrhea,

dementia Still found in some diets that are low

in vitamin content Humans can actually synthesize

nicotinamide from tryptophan; but that’s often in short supply too

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Image courtesy history.nih.gov


Deficiency of thiamine

Beriberi: primary symptoms are in nervous system and musculature

Polished rice is missing thiamine;rice hulls are rich in it



Image courtesy answers.com


Deficiency of cobalamin Available sources of cobalamin are animal

products Therefore vegans need to be careful to get

cobalamin from supplements Symptoms of deficiency (pernicious anemia):

weakness, fatigue, pallor, palpitations, dizziness Deficiency is common even in non-vegans:

5-40% of the population?


Ascorbate

Vitamin in primates, some rodents Synthesizable in most other vertebrates Involved in collagen processing Reduced form acts as reducing agent

during hydroxylation of collagen Deficiency gives rise to inadequate

collagen - scurvy


PTM role of ascorbate(fig. 6.17)

Proline + O2 + -ketoglutarate + ascorbate 4-hydroxyproline + succinate + CO2 + dehydroascorbate

This is a post-translational modification that occurs to prolines within collagen

The hydroxylated prolines help stabilize the collagen triple helix

Hydroxylysine found in collagen too


Dietary deficiency of ascorbate Primary sources of

ascorbate are fruits, particularly citrus, and green vegetables

Ascorbate deficiency’s first symptom involves collagen degradation, leading to scurvy



Image courtesy U.Cincinnati Medical School


Scurvy in history Shortage of green vegetables in

sailors’ diets meant scurvy was rampant on shipboard until the 18th century

Success of English navy over French 1760-1800 was partly due to the introduction of limes in English sailors’ diets 50 years before the French caught on




Lipid vitamins Contain rings & long aliphatic

sidechains At least one polar group in

each Absorbed in intestine, carried

via bile salts Hard to study Most are formally built from

isoprene units, as are steroids


Vitamin A (retinol) 3 forms varying in terminal polar group Involved in signaling and receptors -carotene is nonpolar dimer


Vitamin A deficiency

Produces night blindness because the retina and cornea dry out

Most common cause: nursing infants whose mothers have vitamin A deficiency in their diet


Vitamin D

Several related forms Hormones involved in

Ca2+ regulation

Figure courtesyCyberlipid

(cholecalciferol)


Vitamin D deficiency Rickets in children:

Bone disease, restlessness, slow growth One form of vitamin D is actually

synthesizable from cholesterol given adequate sunlight;

Therefore rickets is most common in densely settled urban environments

Documents

11/11/2008Biochem: Cofactors,Vitamins Cofactors & Vitamins Andy Howard Introductory Biochemistry 11 November 2008