Kinetics of Lactate Dehydrogenase

Unimolecular reactions

A → P or A → P+Q

One reactant w/ 1+ product

Bimolecular reaction

A + B → AB or 2A → A2

A + B → P+Q or 2A → P+Q

2 reactants form 1+ product

Trimolecular reaction

A + B + C → P

3 reactants form 1 product

Modern view of catalysis

Reactant A reacts w/ catalyst E to form EA complex 

Product P is released & original catalyst E is regenerated at the same time

ΔG⁰

Spontaneity of a chemical reaction under standard conditions

-ΔG⁰ = spontaneous reaction & exergonic (produces energy)

ΔG⁰ = 0 means reaction is at equilibrium

How much energy a reaction needs or will produce 

Transition state

High energy & unstable intermediate structure between reactants and products

Peak of activation energy required for a reaction to occur

State that must be passed for the reaction to proceed

Michaelis-Menten kinetics

v = Vmax[S] / Km + [S]

[S] vs v

Km = 1/Vmax

Lineweaver-Burke

1/v0 = Km/Vmax x 1/[S] + 1/Vmax

1/[S] vs 1/v0

x-intercept = -1/Km

y-intercept = 1/Vmax

Sequential reactions

Ordered or random

Dependent on which substrate binds first

Both reactants must be bound to enzyme for reaction to proceed

Group is transferred between the 2 reactants in LDH (sequential ordered)

Ping-pong reactions

One product is released before the second substrate binds

Enzyme is often transiently covalently modified in order to transfer a functional group from one substrate to another

Occurs in serine proteases & aminotransferases

Lactate dehydrogenase (LDH) reaction

Pyruvate + NADH ←→ L-lactate + NAD+

NADH donates hydrogen to pyruvate reducing it to L-lactate

Occurs in anaerobic conditions (no oxygen present)

Oxidoreductase reaction

2 different substrates & 2 different products w/ LDH as enzyme catalyst

LDH isoenzymes

LDH = tetramer (4 subunits)

Subunits can be M or H in mammals w/ different amino acid compositions

5 tetrameric isoenzymes of LDH found in different tissues

LDH-1 = H4 (4 H subunits)

LDH-2 = H3M (3H 1M) - LDH-1&2 found in heart

LDH-3 = H2M2 - lymphatic tissue

LDH-4 = HM3 - LDH-4&5 found in skeletal muscle & liver

LDH-5 = M4 - used in practical

Experimental LDH

Rabbit muscle LDH-5 w/ 4 M subunits

Bovine serum albumin (BSA)

Used to stabilise LDH & keep it in solution & active

Absorbance measurements

Spectrophotometer set to 340

Measuring the disappearance of NADH (reduced to NAD+)

Experiment 2 concept

Doubling the amount of enzyme doubles the rate of reaction

Increasing LDH volume increases ΔA

Experiment 3 concepts

Fixed LDH conc. & fixed NADH at maximum saturation w/ decreasing conc of pyruvate to find Km