Bio-organic Mechanisms Flashcards
Cofactors
organic molecules (coenzymes) or ions (metals) required by enzymes for activity cofactor binds to its apoenzyme (functionally inactive) to form the activated holoenzyme
Coenzyme
low relative molecular mass organic molecules that transfer chemical groups, protons, or electrons
often vitamin derived and behave as cosubstrates
Prosthetic groups
cofactor bound tightly to an enzyme
Vitamins
small organic molecules essential for growth but are unable to be synthesized
Coenzymes in metabolism
- biotin: coenzyme when covalently attached to a carrier protein
- pyridoxine: PLP coenzyme formation
- thiamine: formation of TPP coenzyme
- riboflavin: formation of coenzymse FADH
- niacin: formation of coenzymes NAD/NADP
Coenzymes as electron carriers
NADH/FADH2
- electron transport achieved by proton transfer/2 electrons to these molecules
- coenzymes for redox reactions
FMD/FAD
- link 2 electron and 1 electron transfer reactions
eg. Fe-S clusters can only take 1 electron but NADH give 2, FMN provides a solution to this
Cytochrome
Proteins with heme as prosthetic groups associated with electron transport / redox processes
Covalently attached heme groups (usually with his/met)
Iron ion octahedrally coordinated by 6 ligands
- spectral properties show lower light absorption when oxidiased
Pyruvate Dehydrogenase Structure
- 3 enzymes/5 coenzymes
- connect glycolysis to the TCA cycle via the decarboxylation and oxidation of pyruvate to acetyl CoA
- catalyses C-C bond breakage by stabilising carbanion formation
Breaking C-C bonds
- forms high energy carbanion that needs stabilisation
- ketone group in B position can allow enolate formation for stabilisation (stabilises a position carbanion)
- Schiff base derivative also stabilises as it is an excellent electron sink
- pyruvate bond breakage is difficult, as the ketone group is short 2 electrons and therefore there is no effective stabilisation of the carbanion
Thaimine Pyrophosphate (TPP)
- temporary electron sink
- thiazole ring has partially negative carbon and partially positive nitrogen
- carbanion attacks ketone group
- oxyanion protonates oxyanion
- TPP temporarily bonded to pyruvate
- electrons flow from oxyanion back onto bond, releasing carbon dioxide
- forms Schiff base as an electron sink
Steps of Pyruvate to Acetyl CoA Conversion
- decarboxylation
- oxidation
- transfer to CoA
E. Coli PDH
E1 enzyme - TPP group - oxidative decarboxylation of pyruvate E2 enzyme - lipoamide group - transfer of acetyl to CoA E3 enzyme - FAD group - regeneration of oxidised form of lipoamide
Overview of PDH complex mechanism
- E1 complex decarboxylates pyruvate and links substrate to TPP
- carbanion attacks sulfur bridge of lipoamide to open and reduce it
- acetyl transferred to sulfur of lipoamide
- acetyl transferred to CoA
- reduced form of lipoamide oxidised by E3 (gives up hydrogens)
- E3 gives hydrogens to NAD
E1 Component Mechanism
- carbanion of TPP stabilised by thiazole ring because of the electron withdrawing properties of the S and the positive charge on the N
1. base deprotonates carbon of TPP ring, electron flow back onto carbon (negative charge)
2. carbanion attacks a-keto group of pyruvate - Schiff base stabilises carbanion and allows decarboxylation
3. conjugate acid protonates oxyanion (ketone group) to form hydroxyl
4. negative charged oxygen from carboxyl flows back onto the carbon, releasing carbon dioxide
Coenzyme A
Coenzyme not attached to the enzyme
- forms the final product
- many molecules must be processed
- not regenerated like catalytic coenzymes
- acetyl group added by PDH
E2 Component Mechanism
Lipoamide ‘swinging arm’ is lipoic acid attached to Lysine
- carbanion attacks oxidised sulfur, electron flow onto second sulfur
- base deprotonates hydroxyl, electron flow back onto TPP carbon, so it is removed
- acetylated lipoamide attacked by CoA-SH
Summary of E2
Dihydrolipoyl Transacetylase
- transfer of acetyl group to CoA
- 2 carbon group linked by a thioester bond in both acetylipoamide and acetyl CoA
- thioester is a high energy bond
- lipoamide in reduced form after reaction (sulfurs bound to hydrogens)
E3 Component
Regeneration of lipoamide (oxidised back to disulfide)
- reacts with FAD to remove two electrons/protons
- NAD also reduced to NADH
Coenzyme FAD vs Coenzyme NAD
- FAD linked to E3
- NAD not linked to enzyme, used to regenerate the FAD
E3 Component Mechanism
- check notes**
Coordination of Reactions
PDH enzyme exists in a large complex to allow the coordination of the reactions required for the conversion of pyruvate into acetyl CoA
Lipoamide is the link in the chain of reactions: found on swinging arms moving between E1 and E3
1. allows coordinated catalysis
2. proximity of reaction centers increases rate
3. increases reactant effective concentration
4. intermediates remain tethered
5. minimised side reactions
Lipoamide
- oxidises hydroxyethyl TPP on E1
- transfers acetyl CoA of E2
- regenerated by E3
- movement between subunits to pick up 2 carbon unit, give up 2c unit to CoA, be oxidised/regenerated
Substrate/Metabolite Channeling
Passing of the product of one enzyme directly to another enzyme or active site without its release into solution
Can occur in stable multi enzyme complexes like the PDH complex or in transient assemblies in vivo that form a metabolon
Regulation of PDH
Phosphorylation by PDH kinase inactives enzyme
Dephosphorylation by phosphatase activates enzyme
- kinase is allosterically controlled
- product inhibited by acetyl CoA/NADH
- E1 subunit is target of phosphorylation - kinase actived by the products and inhibited by pyruvate/ADP
Pyruvate Decarboxylase
Ethanol fermentation from pyruvate
TPP coenzyme with carbanion electron sink properties of S/N charges
1. nonoxidative decarboxylation into ethanal
2. reaction with NADH to form ethanol (alcohol DH)
Similar to E1 mechanism of PDH but produces acetaldehyde which is converted to ethanol by ADH
1. hydroxyethyl-TPP deprotonated by base
2. TPP ejected and ethanal goes to ADH
Pyruvate Decarboxylase Mechanism
- glutamine deprotonates TPP, initiating double bond rearrangement resulting in deprotonation of N double bond CH-S group (cofactor activated)
- carbanion of TPP attacks carbonyl carbon of pyruvate in nucleophilic addition that results in the cofactor undergoing double bond rearrangement. glutamine deprotonated
- carbon dioxide eliminated from covalently attached pyruvate intermediate. TPP is an electron sink
- TPP initiates double bond rearrangement, resulting in the asparagine deprotonated, therefore histidine is deprotonated
- glutamine deprotonates TPP, initiating a double bond rearrangement that deprotonates the hydroxide of the intermediate, resulting in the reformation of the carbanionic activated cofactor and the acetaldehyde product
- histidine deprotonates water. carbanion of the TPP cofactor deprotonates the adjacent amine, which initiates double bond rearrangement resulting in glutamine deprotonation
Alcohol Dehydrogenase
Reaction mechanism involves direct hydride transfer of the pro-R hydrogen of NADH to the re face of acetaldehyde
Metal ion + electrostatic catalysis
Pyruvate carboxylase
Oxaloacetate replenishing gluconeogenesis
C-C bond formation
- carbanion attacks partially positive carbon
- ketone group forms enolate electron sink
* **aldol additional reaction
Biotin
Imidazoline ring that is cis-fused to a tetrahydrothiophene ring bearing a valerate side chain
Carboxybiotinyl-enzyme linked via lysine
Carboxylase Mechanism Pt. 1
2 Phase reaction mechanism
- bicarbonate attacks phosphate of ATP, ejecting ADP
- carboxyphosphate formed
- reacts with biotinyl-enzyme
- nitrogen of ring attacks carbon dioxide to load it on
Carboxylase Mechanism Pt. 2
- negative oxygen of carboxyl group on carboxybiotinyl-enzyme flows electrons back to release carbon dioxide
- nitrogen carbon bond attacks pyruvate to pull off hydorgen and form carbon carbon double bond
- biotinyl-enzyme leaves and pyruvate enolate attacks carbon dioxide
- oxaloacetate formed
Citrate Synthase
Formation of citrate from OA and acetyl CoA
Ketone group of OA susceptible to attack
Acetyl CoA forms an enolate (carbanion needed)
1. OA + acetyl CoA gives citroyl CoA
2. hydrolysis of CoA to give citrate
Citrate Synthase Structure
- dimer of identical subunits
- each monomer has 2 domains
- open vs closed conformation
- OA binding induces a large conformational change in the subunits to create the acetyl CoA binding
- sequential ordered kinetics (OA binds first)
- prevents non productive hydrolysis of acetyl CoA
Citrate Synthase Mechanism
- aspartate abstracts proton on acetyl CoA, forms enolate with ketone group that is stabilised by a histidine residue
- OA protonated by histidine (si face)
- electron flow back from enolate onto acetyl CoA
- acetyl CoA attacks OA
- bond formation between CoA and citrate
- hydrolysis (water attacks ketone) at thioester linkage to liberate CoA
Citrate Isomerisation
- citrate isomerized to isocitrate
- citrate is a tertiary alcohol so cannot form a carbonyl via oxidation
- isocitrate is a secondary alcohol that can be oxidised
Isomerisation Mechanism
Catalysed by aconitase
- elimination reaction of water
- cis-aconitate intermediate with carbon double bond
- addition of water to a different water to from isocitrate
Aconitase Structure
Fe-S cluster used to orient substrate in active site
3 cys ligate, 1 free ion to ligate substrate
- active form adds final iron ligand
- stereospecificity in which of the prochiral hydrogens are removed from the C2
Aconitase Mechanism
- base deprotonates citrate and histidine pulls off water
- carbon carbon double bond formed in intermediate
- molecule flips
- water re-added and base is deprotonated to add water back
- stereospecific addition of water to double bond to re face
Isocitrate DH
Catalyses the oxidative decarboxylation of isocitrate to a-ketoglutarate
- oxidation to oxalosuccinate
- decarboxylation of B-keto acid to form a-ketoglutarate
A-ketoglutarate oxidative decarboxylation
Second redox reaction of TCA cycle
Removes carbon dioxide, oxidises resulting molecule, adds it as an acyl group to CoA to form succinyl CoA
Reaction resembles the PDH complex one
Comparison of Oxidative decarboxylations
Pyruvate vs a-ketoglutarate
- both substrates a-ketoacids
- product is a thioester with CoA
Catalysed by homologous complexes with analogous reaction mechanisms
Succinyl CoA Synthetase
- succinyl CoA to succinate
- break high energy thioester bond to form high energy phosphate bond of ATP/GTP
Regeneration of OA from succinate
- oxidation of methylene to an unsaturated bond
- hydroation of a double bond to hydroxyl
- oxidatoin of hydroxyl to carbonyl
Energy abstracted in form of high energy electrons
Elimination Reactions
E1:
- base deprotonates carbon, and electrons flow onto leaving group in concerted mechanism
E2:
- leaving group leaves to form carboanion
- base deprotonates carbon to form double bond
E1cB:
- base deprotonates to form carbanion
- electrons then flow onto leaving group in RDS
If carbanion can be stabilised the E1cB will be removed, such as having an electron sink
Succinate Elimination
E1 not plausible due to the high energy intermediate
E1cB mechanism uses the carboxyl group ketone to form enolate stabilising carbanion (resonance)
Fumarase
Stereospecific enzyme : only one isomer of malate formed from fumarate
Catalyses hydroxyl addition to the double bond
- need base to make water nucleophilic
- double bond attacked by nucleophile
- acid protonates carbanion
- E1cB mechanism for elimination of water from malate to form fumarate
Malate DH
Final step in TCA cycle
- NAD is electron acceptor
- significant positive free energy so the reaction is driven by product consumption
Succinate DH structure
- prevents ROS generation with heme groups (heme b)
- flavin or FADH can produce ROS like HO2, or H2O2
- heme is an emergency electron sink
- electrons can do down the chain rather than allowing flavin reaction with molecular oxygen
Pyridoxal Phosphate
- key enzyme in aminotransferase reactions
- all amino acids (minus glycine) are chiral
- carbon backbone comes from glycolytic pathway/pentose phosphate/TCA cycle
- N comes from ammonia and is introduced stereospecifically by aminotransferases
Aminotransferase Reaction
Net: aa 1 + a-ketoacid 2 –> a-ketoacid 1 + aa 2
Amine transferred from aa 1 to a-keto acid 2, which becomes aa 2
- glutamate is usually amine group donor
Features of PLP
- reactive aldehyde
- acidic phenolic group
- basic pyridine ring with N that can be protonated (electron sink)
- H bonding possible between H of aldehyde and O
Stable tautomeric forms allow PLP to act as an electron sink during reactions
Glutamate
Can be synthesized from a-ketoglutarate and ammonia de novo
Other chiral amino acids are synthesized with the correct stereochemistry at the a-carbon by stereospecific transfer of the amino group from glutamate
Aminotransferase 2-step Reactions
- aa 1 + enzyme PLP –> a-keto acid 1 + enzyme -PMP
amine transferred from aa 1 to pyridoxal group - a-ketoacid 2 + enzyme PMP –> aa 2 + enzyme PLP
amine transferred from pyridoxamine group to a-keto acid 2
amino acid 2 is produced and PLP regenerated
Group transfer reaction
Covalent catalysis with Ping Pong mechanism
- aa 1 binds first and transfers group to PLP, making it PMP
- a-keto acid 1 leaves and a-keto acid 2 binds
- group transfer to a-keto acid 2, which leaves as aa2
Transimination Reaction
- enzyme PLP schiff base formation (covalent linkage)
- lysine has primary amine functionality to form Schiff base with PLP - amine on aa attacks Schiff linkage
- proton leaves and enzyme ejected
- amino acid PLP schiff base formation (aldimine)
- h bonding between PLP ring and substrate fixes molecule’s conformation
Tautomerization
- deprotonation by lysine
- electron flow around PLP ring to stabilise negative charge of alpha carbon
- Schiff base double bond switches - lysine deprotonated
Hydrolysis
Forms pyridoxamine phosphate and a-keto acid released
**see notes for further mechanism explanation*
Aminotransferase Mechanism Summary
- Schiff base formation
- Tautomerization
- hydrolysis to produce first keto-acid
- PLP carrying amino group left
- a-keto acid comes in and forms Schiff base
- second tautomerization
- hydrolysis to release amino acid
Planarity of the Intermediate
Important to resonance stabilisation
Planarity of molecule essential for pi orbital overlap
H bonding prevents rotation to keep molecular planar
Carboxylate of a-carbon held in place by coordination to an arginine
Proton being transferred can only come from one direction so there is only one stereoisomer being produced
Selection of Bond for Cleavage
Elimination occurs perpendicular to the plane of the delocalised system
Scissile bond kept in place
Decarboxylation
- Schiff base formation
- carboxylate group removed and electron movement around ring
- protonation of carbanion by acid
- Schiff hydrolyzed
Racemization
- base deprotonates amino acid
- electron flow around ring
- electron flow around ring back the way it came
- second base reprotonates but with different stereospecifically
- Schiff base hydrolyzed to free amino acid
