1

How much enzyme have I got?

Enzyme “activity”
• Measure by rate of reaction catalysed
(substrate disappearance and/or product
increase)
• For amount of enzyme need rate in terms of
amount
– Commonly use either mol min-1 (“international
units” or “IU”) or mol s-1 (“katals” or “kat”)
 2

How active is my enzyme?

“specific activity”
• Need another measure of quantity
- weight of purified enzyme or total protein
• Specific activity = activity divided by weight of
protein
– e.g. mol min-1 mg-1 (= IU mg-1)
or mol s-1 kg-1 (= kat kg-1)
 3

ENZYME KINETICS

• How does reaction rate depend on

concentration of substrates, enzyme, other
compounds?
 4

Typical progress of reaction with time

• Starts with a linear section, giving “initial rate”
• Followed by a slowing down, possible causes
– Substrate concentration falls
– Inhibition by accumulating product
– Inactivation of enzyme
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Effect of enzyme concentration on rate

• Normally rate is directly proportional to

enzyme concentration
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Effect of substrate concentration: saturation

kinetics
• In most simple chemical reactions, rate is proportional
to some power of reagent concentration - often linear,
“first order”
• Nearly all enzyme-catalysed reactions are more
complicated
First order
reaction Typical enzyme
Rate
reaction

0
0 [Substrate]
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Saturation kinetics occur because enzyme
forms a complex with substrate before
reaction is catalysed

• At low [substrate], many enzyme

molecules do not have a substrate
molecule bound (at any one time)
• At high enough [substrate], all enzyme
molecules have bound substrate, and are
working as fast as they can
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THE MICHAELIS-MENTEN EQUATION

For the mechanism:
k1 k2
E + S ES E + P
k1
d [ P] d [ S ] k 2 [ E ]o [ S ] Vmax [ S ]
v   
dt dt ( K m  [ S ]) ( K m  [ S ])
[ E ]o is total enzyme concn ( [E]  [ES])
k 2 [ E ]o  Vmax (maximum rate at high [S])
K m is " Michaelis constant"
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Some key features of the shape of the Michaelis-

Menten graph
• When [S] >> Km v Vmax (or Vm)
• When [S] = Km v = Vm /2
• When [S] << Km , rate increases linearly with [S]
v  (Vm/Km)*[S]
Vm

Vmax [ S ] Vm/2
v
( K m  [ S ]) Km
0 [S]
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Units for v and Vm

• Derivation above makes them rates of
concentration change (e.g. mM s-1)
• But equation works for other types of units,
provided use same ones for v and Vm. Can
use rate of:-
– amount transformed e.g. mol min-1
– amount per unit enzyme e.g. mol min-1mg-1
– change in experimental measure, e.g.
absorbance change, titrant addition in pH stat
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kcat
The rate constant of the catalytic step in the
active site
From:-
• specific activity of pure enzyme saturated
with all substrates
• and its molecular weight
calculate as mol reaction (mol enzyme) -1
(time) -1, or just e.g. s-1
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THE SPECIFICITY CONSTANT, kcat/Km

• Slope of rate against substrate

concentration at low [S]
• Determines selectivity between competing
substrates
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ENZYME INHIBITION

• Many compounds can reduce the rate of

enzyme reactions
• These enzyme inhibitors are important
– in metabolic regulation
– as drugs
– as the origin of toxicity
– in the study of enzyme mechanisms
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ENZYME INHIBITORS

• Reversible - there is an equilibrium between

enzyme with bound inhibitor and the free
molecules
• Irreversible - once an inhibitor is bound, then
enzyme is affected permanently
• Usually reversible inhibitors bind non-
covalently, irreversible covalently
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COMPETITIVE REVERSIBLE INHIBITORS

• An important class of inhibitors compete with

the substrate to bind at the active site
• Often they resemble the substrate in shape,
charge etc.
• At sufficiently high [S], the substrate will always
win the competition, so Vmax is unchanged
• At fixed inhibitor concentration, will find
apparent increase in Km