TDC2h

Enzymes as machines
October, 2020
 Outline

Enzymes as molecular jigs

PGK as an example

Mechanism

Structural Data

PGK in cells
Jigs
Jigs and Molecular Jigs

• Align things to make them easier to work on

• Protect the components so they are processed

correctly

• Does not need an energy source

• Can be used again after process completed

 Outline

Enzymes as molecular jigs

PGK as an example

Mechanism

Structural Data

PGK in cells
PGK - location
 Reactants

Glycerol

3PGA
3-Phosphoglyceric Acid 1,3 PGA
3-Phosphoglycerate
1,3-Phosphoglyceric Acid
1,3-Phosphoglycerate
1,3-bis-phosphoglycerate
 Reaction

1,3 PGA ADP

3 PGA ATP
 Reaction

O
O
P
O O
O O 3PGA
O
1,3 PGA O
HO O
O P
O HO O P O
O
O
 Reaction

ADP ATP
O
O
P
O O
O O 3PGA
O
1,3 PGA O
HO O
O P
O HO O P O
O
O
 Reaction

ADP ATP
O
O
P
O O
O O 3PGA
O
1,3 PGA O
HO O
O P
O HO O P O
O
O
 Reaction

AMP
O AMP
O
ADP O P O
O P O ATP
O
O
O P O
O O
O
P
O O
O O 3PGA
O
1,3 PGA O
HO O
O P
O HO O P O
O
O
 Outline

Enzymes as molecular jigs

PGK as an example

Mechanism

Structural Data

PGK in cells
 PGK -reaction
ATP
3PGA
1,3PGA

1,3PGA
1,3 PGA 3PGA
1,3 PGA

ADP ATP

ADP
3PGA ATP
 Test direct transfer
hypothesis

ADP

High [ADP]
V

[1,3 PGA]
Much same result if you use high 1,3PGA and vary ADP
Jigs and Molecular Jigs

• Align things to make them easier to work on

 In-line attack?
1,3-PGA ADP
O-
O AMP
P O- P
O O
O-
O O-
3-PGA

3-PGA ATP
In line attack
1,3-PGA ADP
O-
O AMP
P O- P
O
O- O
O O-
3-PGA
In line attack
1,3-PGA ADP
O-
O AMP
P O- P
O
O
O O -
O-
3-PGA
 In line attack
ADP

O AMP
O- O- P
P O
O-
3-PGA O O-

Inversion of configuration
 In-line? Evidence?
1,3-PGA ADP
S O AMP
17
O- P
P O
O 18
O- O-
O
3-PGA

3-PGA ATP
In-line mechanism -probable
ATP

S O AMP
17
O P
P O
O O-
3-PGA +
18
O-
Inversion of configuration

1,3 PGA
ADP
No Enzyme-Pi complex
Pi Pi ADP
-double inversion
3 PGA
 Design for our enzyme
Align components precisely

Reduce activation
energy

Protect from water

 Design for our enzyme
Align components precisely
 Design for our enzyme
Reduce activation
energy
 Design for our enzyme
Protect from water
Easily
hydrolysed

H
O
H
 Outline

Enzymes as molecular jigs

PGK as an example

Mechanism

Structural Data

PGK in cells
Crystal Structure
Crystal Structure

3PGA
Crystal Structure

ADP
Crystal Structure

ADP3PGA
Model Transition state
Lys 219

3PGA

ADP
Mg2+ Arg 39
Before Conformational Change
Lys 219

3PGA

ADP
Mg2+ Arg 39
Transition state
1,3-PGA ADP
O-
O AMP
P O- P
O
O- O
O O-
3-PGA
Transition state
1,3-PGA ADP
O-
O AMP
P O- P
O
O
O O -
O-
3-PGA
 Transition state
ADP

O AMP
O- O- P
P O
O-
3-PGA O O-
 Transition state
+ ADP
O-
O AMP
P O- P
O
O
O O -
O-
3-PGA +

Closes only when both substrates - avoids hydrolysis

 Design for our enzyme
Align components precisely

Reduce activation
energy

Protect from water

Where is energy released?

ATP
3PGA
1,3PGA

1,3PGA
3PGA
ADP ATP

ADP 3PGA ATP

?

DG0=-18.8kJ.mole-1
Enzyme transfer is at
equilibrium

DG  0
E.1,3PGA.ADP E.3 PGA.ATP
 Enzyme transfer is at
equilibrium

DG  0
E.1,3 PGA.ADP E.3 PGA.ATP

How can this be?

Where then is the energy released?

But you answer on the Seeded Question on the

Discussion Board. Link
Release of products is rate
limiting

DG  0
E.1,3 PGA.ADP E.3 PGA.ATP E
+
3 PGA
+
ATP
Negatively
charged products
released
Release of products related
to conformation change

DG  0
E.1,3 PGA.ADP E.3 PGA.ATP E
+
3 PGA
+
ATP
 Outline

Enzymes as molecular jigs

PGK as an example

Mechanism

Structural Data

PGK in cells
PGK in Cells

Crowded - limited diffusion

Why doesn’t 1,3PGA get hydrolysed

before it finds PGK?
 PGK in Cells
[PGK] 130mM
[GAPDH] 1400mM
1,3 PGA
1,3 PGA 50mM
3 PGA 200mM
PGK GADPH
High concentration of E vs S

So all S is enzyme bound

PGK and TIM; a complex?
Fusion between PGK and TIM-
Hyperthermophile - Thermotoga maritima

PGK

TIM
 Alice and the Red Queen

"Alice laughed: "There's no use

trying," she said;
"one can't believe impossible things.“

"I daresay you haven't had much

practice," said the Queen.

"When I was younger, I always did it

for half an hour a day. Why,
sometimes I've believed as many as
six impossible things
before breakfast."
 Summary
Enzyme is a molecular jig:
aligns
facilitates
reuseable

Conformational change only trips reaction

when both substrates bound

Glycolytic Enzymes are often present at

as high (or higher) concentration than
substrate. Thus intermediates may never
freely diffuse in solution