9: HEXOKINASE AND LACTATE DEHYDROGENASE

Question 1

Hexokinase -example of induced fit

Answer 1

• the residues in the active site envelop the S
• then get good catalysis because we’re changing the microenvironment and moving the residues in the active site to the appropriate location to catalyse the reaction
• binding of S induces a change in conformation of enzyme from open to closed state

Question 2

Hexokinase - mechanism

Answer 2

• First step of glycolysis: Phosphorylation of Glu to Glu-6-P
  1. Nu attack using LP on C6-OH of Glu on the y-phosphate of an Mg2+-ATP complex (e.g. metal ion catalysis)
  2. in phosphorylation reaction, phosphoryl group is transferred
  3. P=O is a polarised system; P is an electrophile so its susceptible to nucleophilic attack (SN2-type)
  4. B in concerted mech. pulls off proton from O to promote nucleophilicity
  5. leaving group is ADP, transfer phosphoryl to O to form phosphorylated Glu

Question 3

Why does ATP react w/OH in Glu but not in water? (Hexokinase)

Answer 3

• competing side reaction
• use induced fit mechanism
• hexokinase has 2 lobes for flexibility
• once ATP and Glu bind into active site, 2 lobes move and snap shut to bury 2 substrates
• it drives water out from the active site so in closed conformation is relatively dry
• so when we line up ATP and Glu for reaction, there’s no interfering after molecules to go in and compete w/phosphoryl group transfer

Question 4

Lactate dehydrogenase reaction

Answer 4

• e.g. example of stereospecific formation of lactate from pyruvate (a-keto-acid)
• pyruvate reduced using NADH
• reversible reaction

Question 5

Lactate structure

Answer 5

• sp3 chiral centre

- S and R-lactate enantiomers can be formed in a non-enzyme catalysed reaction

Question 6

lactate dehydrogenase reduction mechanism

Answer 6

1. Nu attack of H- (hydride) to C of ketone (concerted mechanism)
2. forms O- but is rapidly protonated to form secondary alcohol and conj. base

Question 7

lactate dehydrogenase formation of single stereoisomer

Answer 7

• hydride transfer from only one direction
• have to pin pyruvate into active site so that only one face of that trig. planar molecule is accessible to H-
• carboxylate group forms v.strong charge-charge interactions w/protonated Arg side chain
• H-bonded between +ve Arg and His side chain w/carbony-keto-oxo group (interactions ensure pyruvate to fit into particular geometry)
• hydride is attacking re face; coming down from above
• only pro-R H is transferred
• have free rotation about R-group (rest of NADH) and N so ring can twist around; interaction between 1º amide and residues in active site that pin the ring in particular orientation so that’s fixed and only pro-R can work

Question 8

chiral centre definition

Answer 8

• atom in molecule bonded to 4 different chemical species

- allows for optical isomerism

Question 9

prochiral centre definition

Answer 9

-when molecule can be converted to a chiral center by changing only one of the attached groups

Question 10

lactate dehydrogenase mechanism

Answer 10

1. H- carried by nicotinamdie ring
2. 2e- in bond are transferred and perform nucleophilic attack on C centre
3. break octet rule around C
4. 2e move out from pi-bond
5. His195 acts as gen.acid to protonate oxyanion species so we end up w/lactate

Question 11

R and S isomers

Answer 11

-R higher priority groups clockwise

Question 12

how does enzyme ensure stereospecificity

Answer 12

• enzymes can recognise pro-chiral centre by binding S, they fix the molecule in space so only one H can be transferred to one face of another group so that we end up w/100% production of particular stereoisomer not a racemic mixture
• CH3 and OH groups are recognized by the enzyme and they bind into the enzyme, and because of the tetrahderal nature of the C-centre it means that the pro-S H binds into the active site leaving the pro-R H free and accessible -due to geometric and electronic complementarity