27-28: BIOORG MECHS

Question 1

citrate synthase

Answer 1

1. acetyl coa + oxaloacetate = citrate
   - in mammals, enzyme is a dimer of identical subunits
   - exists in open/close conformation
   - binding of oxaloacetate induces large conformational change which creates binding site for acetyl coA
   - sequential ordered kinetics: oxalo binds, then acetyl CoA, then reaction is catalysed; this prevents non-productive hydrolysis of acetyl CoA

-claisen condensation

Question 2

citrate synthase mechanism

Answer 2

• to form enolate, have to deprotonate acetyl CoA by Asp375 (B); stabilised by H-bond
• Nu attack on si face of oxaloacetate (stereospecific); generate -ve His320 side chain
• form S-citry-CoA; hydrolysis by water to liberate CoA

Question 3

isomerisation citrate to isocitrate

Answer 3

• citrate is pro-chiral; 3º alcohol so cannot be oxidised
• isomerisation to convert to 2º alcohol
• anti elimination of water to produce cis-aconitate; then anti-addition of water back on different atoms
• aconitase enzyme is a lyase as it goes through an intermediate which can detach from the enzyme and re-bind; 2 back-to-back lyase reactions; elimination reaction where water is being removed

Question 4

aconitase FeS clusters

Answer 4

• enzyme contains an FeS cluster, not in redox role, but to orient substrate in the active site; ligated by 3 Cys residues
• Fe in cluster has ligand positions available to bind in the S and water molecule; acting as lewis acid to accept LP of e; His101 and Ser642 residues

Question 5

oxidative decarboxylation of isocitrate

Answer 5

-intermediate is oxalosuccinate (ß-keto-acid)

Question 6

oxidative decarboxylation of a-ketoglutarate

Answer 6

• analogous to PDH complex reaction
• has E1/2/3/ enzymes; E1 a-ketoglu DH; E2 transsuccinylase; E3 dihydrolipoyl DH
• remove CO2; add CoA; NAD+ reduced to NADH

Question 7

regeneration of oxaloacetate from succinate

Answer 7

-oxidation/hydration/oxidation: 3 reactions constitute a metabolic motif also seen in FA synthesis/degradation and degradation of some AAs

Question 8

E1cB mechanism

Answer 8

• E1 mechanism (elimination) going through conjugate base
• B removing proton step comes first (fast step)
• RDS is ejection of leaving group from conj.B

Question 9

succinate DH complex

Answer 9

• elimination reaction
• hydrophilic domain where succinate binds
• heme b plays a role in preventing the formation of reactive oxygen species ROS (side reaction that occurs); emergency e sink
• flavins (FADH/FADH2) are weak links in ETC; they produce ROS that lead to damage

Question 10

fumarase

Answer 10

• malate is a chiral molecule
• anti addition
• S stereoisomer
• OH- attacks double bond
• reverse reaction is E1cB

Question 11

malate DH

Answer 11

-hydride transfer

Question 12

amino acids

Answer 12

• all chiral except glycine
• C backbone comes from glycolysis/pent-5/TCA pathway
• N derived from ammonia
• N introduced stereo-specifically by aminotransferases

Question 13

pyridoxal phosphate

Answer 13

• derived from pyridoxine vit B6
• basic pyridine ring; N has LP available for protonation; good e sink
• aldehyde group; can react w/amino group to form schiff base
• hydroxy group attached to ring (acidic phenolic group that can H bond); can deprotonate as has low pKA; forms phenolate anion

Question 14

aminotransferase reaction

Answer 14

• amine transferred from AA1 to pyridoxal group to form pyridoxamine phosphate
• amine transferred to a-ketoacid2; AA2 is produced; PLP is regenerated
• example of group transfer reaction via ping-pong mechanism; involves covalent catalysis

Question 15

aminotransferase mechanism

Answer 15

1. TRANSIMINATION
   - amino acid can replace lys side chain of enzyme-PLP schiff base; transfer PLP; eject protonated lys side chain; gives AA-PLP schiff base (aldimine); can deprotonate phenol group; H bond keeps molecule planar
2. TAUTOMERISATION
   - aldimine appropriately positioned in active site through interactions w/+ve Arg side chains; specific B pulls off proton (only works if carbanion can be stabilised); forms intermediate; reprotonate; end up w/tautomer called ketimine
3. HYDROLYSIS
   - results in a-ketoacid
   - form pyridoxamine phosphate
4. 2nd substrate a-ketoacid comes in to convert it to a new AA; forms schiff base; second tautomerization reaction; hydrolysis to release second product

Question 16

aminotransferase reaction - how is stereochemistry controlled?