Chem 109 C Bioorganic Compounds

Fall 2019 HFH1104

Armen Zakarian Office: Chemistry Bldn 2217 http://labs.chem.ucsb.edu/~zakariangroup/courses.html

CLAS Instructor: Dhillon Bhavan [email protected] Chapter 24: Coenzymes

2 FAD-FADH2, FMN-FMNH2

vitamin B2: NH2 NH2 N N N N OH O O N N O O N N H OH P P O P P O O O O O O O H OH O- O- O- O- H OH H OH H OH HO OH HO OH H OH H OH CH2 H OH H OH N N O CH CH 2 2 NH H N N N O N N O O NH NH riboflavin N N H O O

FAD FADH2

• catalyze redox (oxidation-reduction) reactions • source of riboflavin: milk, liver, mushrooms, etc. • deficiency disease: skin inflammation, cracked lips, sensitivity to light… FAD-FADH2, FMN-FMNH2

NH2 N N

O O O N N P P P O O O- O O O O- O- O- H OH H OH HO OH NH2 H OH H OH N H OH H OH N CH 2 CH2 O O O N N N N O N N O P P P O -O O O O - - - NH NH O O O N N HO OH O O adenosine triphosphate FMN FAD

PRACTICE PROBLEM FAD is obtained by an enzyme-catalyzed reaction that uses FMN and ATP as substrates. What is the other product of the reaction? FAD-FADH2, FMN-FMNH2

Examples of reactions catalyzed by FAD or FMN:

dihydrolipoyl HS SH dehydrogenase S S - - O + FAD O + FADH2

O O

D- or L-amino acid O O oxidase R - R O + FAD O- + FADH2 NH2 NH

succinate O O - dehydrogenase - O - O - O + FAD O + FADH2 O O

NADH dehydrogenase + + NADH + H + FMN NAD + FMNH2 FAD-FADH2, FMN-FMNH2

dihydrolipoyl HS SH dehydrogenase S S - - O + FAD O + FADH2

O O

D- or L-amino acid O O oxidase R - R O + FAD O- + FADH2 NH2 NH

succinate O O - dehydrogenase - O - O - O + FAD O + FADH2 O O

NADH dehydrogenase NADH + H+ + FMN NAD+ + FMNH mechanism is on page 1073 2 (and discussed in class) FAD-FADH2, FMN-FMNH2

dihydrolipoyl HS SH dehydrogenase S S - - O + FAD O + FADH2

O O

D- or L-amino acid O O oxidase R - R O + FAD R O- + FADH2 NH2 1 NH N 10a N O succinate O O - dehydrogenase NH - O - O - O + FAD N 4a O + FADH2 5 O O O

NADH dehydrogenase + + NADH + H + FMN NAD + FMNH2 PROBLEM 4 Instead of adding the the 4a position and protonating N5, the thiolate ion could have added to the 10a position and protonated N1. Why is addition to the 4a position favored? FAD, FMN, FADH2, FMNH2 PRACTICE PROBLEM [homework]

Propose a mechanism for the reduction of lipoate by FADH2 S S HS SH - - O + FADH2 O + FAD

O O

:B H-B R R H N N O -O N N O O NH NH N N H H O O S FADH2 B H S

R R N N O N N O

NH NH N N

- H O O B: HS S B H - O- HS S O- O O8 FAD, FMN, FADH2, FMNH2

Oxidation of α-amino acids to α-imino acids

D- or L-amino acid O O oxidase R - R O + FAD O- + FADH2

NH2 NH

R H B R H N N O N N O R C COOH- + NH NH NH N N H O O

H H B

R C COO-

NH2

B: Chapter 23: Coenzymes

Coenzyme Vitamin Reaction catalyzed NAD+, NADP+ / NADH, oxidation/reduction of niacin, nicotinamide NADPH alcohols oxidation/reduction, FAD / FADH riboflavin (B2) 2 other Thiamine pyrophosphate thiamine (B1) acyl group transfer TPP Lipoic acid /dihydropipoic lipoic acid oxidation/reduction acid Coenzyme A, CoASH pantothenic acid (B5) acyl group transfer

Biotin biotin (B7) carboxylation

Pyridoxal phosphate PLP pyridoxin (B6) 6 amino acid reactions

Coenzyme B12 vitamin B12 isomerization

Tetrahydrofolic acid, THF folic acid one-carbon transfer

Vitamin KH2 vitamin K carboxylation

Vitamin KH2 is not soluble in water see Table 23.1 in Chapter 23 FAD, FMN, FADH2, FMNH2 PRACTICE PROBLEM [homework]

Propose a mechanism for the reduction of lipoate by FADH2 S S HS SH - - O + FADH2 O + FAD

O O FAD, FMN, FADH2, FMNH2 PRACTICE PROBLEM [homework]

Propose a mechanism for the reduction of lipoate by FADH2 S S HS SH - - O + FADH2 O + FAD

O O

:B H-B R R H N N O -O N N O O NH NH N N H H O O S R FADH2 1 B H S N 10a N O

NH R R N 4a 5 O N N O N N O

NH NH N N

- H O O B: HS S B H - O- HS S O- O O FAD, FMN, FADH2, FMNH2

Covalent attachment of FAD to Cys [or His]

R R

N N O Base N N O H+ NH NH N N O O FAD

R SCys R R H H SCys N N O CysS N N O oxidation N N O NH N H+ NH NH N N O H O O E-FAD

PROBLEM 7 Why the hydrogens of the C8 methyl are more acidic than those at the C7 methyl? Pyruvate dehydrogenase complex

pyruvate dehydrogenase O system O O- + CoASH + CO SCoA 2 O TPP, lipoate, CoASH, FAD, and NAD+ Thiamine pyrophosphate, TPP

H vitamin B1: NH2 O O + N S P P N - O - O - O O O NH2 N N+ S thiamine pyrophosphate, TPP N OH N

resonance structure thiamine

• catalyze two-carbon transfer, many reactions in catabolism • source of thiamine: flax, oatmeal, potato, liver, eggs, etc. • deficiency disease: peripheral NS, beriberi TPP

pyruvate decarboxylase O TPP O O- + H+ + CO H 2 O

! thiazole is relatively acidic ! ylide carbanion is a good nucleophile ! and, as a fairly week base, a good leaving group Lipoate-Dihydrolipoate

vitamin:

S S H N S S Lys OH O lipoate O lipoic acid

redox

• redox/part of pyruvate dehydrogenase system, aerobic catabolism • source of lipoate: almost all foods • deficiency disease: none Coenzyme A (CoASH)

O O O O vitamin B5: O O SH O O S P N N P N N - H H - H H O OH O OH O O O NH NH HO O 2 O 2 N OH H N N N N OH O O P N N P N N pantothenic acid -O O O -O O O

O OH O OH P O P O -O -O -O -O CoASH acetyl-CoA

• acyl transfer, part of PDH system, pyruvate metabolism • source of pantothenic acid: almost all foods, but more in meats • deficiency disease: extremely rare, similar to starvation TPP

pyruvate decarboxylase O TPP O O- + H+ + CO H 2 O

OH O- O B H CH3 C O + O C O R + O- N S B H

O OH OH

C CH3 C CH3 C R N+ S C C R N+ S R N S

:B O H

CH3 CH O + C R H N+ S Pyruvate dehydrogenase system

pyruvate dehydrogenase O system O O- + CoASH + CO SCoA 2 O TPP, lipoate, CoASH, FAD, and NAD+

H B CONHEnzyme2 - S :B S OH OH O H

CH3 C CH3 C CH3 C S SH C C C NHEnzyme2 R R N+ S N S R N+ S O

O CoASH CH C S SH SH SH O 3 + NHEnzyme2 NHEnzyme2 CH C SCoA + 3 C O R N+ S O FAD-Enzyme3

NAD+ S S FAD-Enzyme3 FADH -Enzyme3 2 + NHEnzyme2 (-NADH, -H+) 20 O Pyruvate dehydrogenase complex

pyruvate dehydrogenase O system O O- + CoASH + CO SCoA 2 O TPP, lipoate, CoASH, FAD, and NAD+

H B CONHEnzyme2 - S :B S OH OH O H

CH3 C CH3 C CH3 C S SH C C C NHEnzyme2 R R N+ S N S R N+ S O

O CoASH CH C S SH SH SH O 3 + NHEnzyme2 NHEnzyme2 CH C SCoA + 3 C O R N+ S O FAD-Enzyme3

NAD+ S S FAD-Enzyme3 FADH -Enzyme3 2 + NHEnzyme2 (-NADH, -H+) O Pyruvate dehydrogenase complex

pyruvate dehydrogenase O system O O- + CoASH + CO SCoA 2 O TPP, lipoate, CoASH, FAD, and NAD+

H B CONHEnzyme2 - S :B S OH OH O H

CH3 C CH3 C CH3 C S SH C C C NHEnzyme2 R R N+ S N S R N+ S O

O CoASH CH C S SH SH SH O 3 + NHEnzyme2 NHEnzyme2 CH C SCoA + 3 C O R N+ S O FAD-Enzyme3

NAD+ S S FAD-Enzyme3 FADH -Enzyme3 2 + NHEnzyme2 (-NADH, -H+) O Pyruvate dehydrogenase complex

pyruvate dehydrogenase O system O O- + CoASH + CO SCoA 2 O TPP, lipoate, CoASH, FAD, and NAD+

H B CONHEnzyme2 - S :B S OH OH O H

CH3 C CH3 C CH3 C S SH C C C NHEnzyme2 R R N+ S N S R N+ S O

O CoASH CH C S SH SH SH O 3 + NHEnzyme2 NHEnzyme2 CH C SCoA + 3 C O R N+ S O FAD-Enzyme3

NAD+ S S FAD-Enzyme3 FADH -Enzyme3 2 + NHEnzyme2 (-NADH, -H+) O practice problem

PRACTICE PROBLEM Acetolactase synthase can also transfer the two-carbon fragment from pyruvate to α-ketobutyrate, forming α-aceto-α-hydroxybutyrate. This is the first step in the formation of isoleucine. Propose a mechanism for this reaction.

acetolactase synthase O O TPP O OH O- + COO- + CO O- 2 O O Biotin

attaches to Lys by forming an amide O vitamin H or B7: HN NH

OH biotin S (itself) O biotin

• carboxylation (aldol with CO2) • source of biotin: liver, soybeans, tomato, carrots • deficiency: rare and mild, skin conditions, perosis, FLKS Biotin

attaches to Lys by forming an amide O vitamin H or B7: HN NH

OH biotin S (itself) O biotin

pyruvate carboxylase ATP, Mg2+ O O O O biotin O 2- O + O + ADP + HPO4 HO O O O

pyruvate oxaloacetate

acetyl CoA carboxylase ATP, Mg2+ O O O O biotin + ADP + HPO 2- SCoA + O SCoA 4 HO O

acetyl CoA malonyl CoA Biotin

acetyl CoA carboxylase ATP, Mg2+ O O O O biotin + ADP + HPO 2- SCoA + O SCoA 4 HO O acetyl CoA malonyl CoA

O O O O P O HO O O O O O O N NH Mg unreactive as much better electrophile electrophile S R

key intermediate in biotin-catalyzed carboxylations O base O

R R Biotin