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Spring 2013 Lecture 15.pdf

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CHM333 LECTURES 15: 2/20/13

SPRING 2013

Professor Christine Hrycyna

ENZYME KINETICS:
•

The rate of the reaction catalyzed by enzyme E
A+B↔P
is defined as
-Δ[A] or
Δt
•
•

-Δ[B] or
Δt

Δ[P]
Δt

A and B changes are negative because the substrates are disappearing
P change is positive because product is being formed.

•

Enzyme activity can be assayed in many ways
– disappearance of substrate
– appearance of product

•

For example, you could measure
– appearance of colored product made from an uncolored substrate
– appearance of a UV absorbent product made from a non-UV-absorbent substrate
– appearance of radioactive product made from radioactive substrate
•

•

Many other ways possible – Just need a way to distinguish the
products from the substrates

The VELOCITY (reaction rate) (product formation of disappearance of substrate/time)
of an enzyme catalyzed reaction is dependent upon the substrate concentration [S].
Velocity related to [S]

Enzyme Kinetics: Velocity
The velocity (V) of an enzyme-catalyzed reaction is
dependent upon the substrate concentration [S]

•  A plot of V vs [S] is often hyperbolic
Michaelis-Menten plot
Graph is not a graph of product formation over time!!!
104

CHM333 LECTURES 15: 2/20/13
•

SPRING 2013

Professor Christine Hrycyna

An example of how to do a kinetics experiment:
A.Take 9 tubes, add identical amount of enzyme (E) to each tube
B. Each tube contains an increasing amount of substrate (S) starting with zero
C. Measure the velocity by determining the rate of product formation
D.Plot these values – Velocity against substrate concentration
E. Generate the curve shown:
i. Often the shape is hyperbolic – a characteristic of many enzymes – shape
suggests that the enzyme physically combines with the substrate – ES
complex
ii. Called a SATURATION PLOT or MICHAELIS-MENTEN PLOT after
the two biochemists that first described and explained the curve shape.

•

Let’s look at the various features of the plot:

A. As [S] is first increased, the initial rate or velocity (V0) increases with increasing
substrate concentration
i. V is proportional to [S]
B. As [S] increases, V increases less and less
i. V is NOT proportional to [S] in this range
C. Finally, V doesn’t increase anymore and velocity reaches its maximum (Vmax)
i. Enzyme is working as fast as it can
D. Velocity won’t change no matter how much substrate is present. At this point, the
enzyme is saturated with substrate, S.

105

CHM333 LECTURES 15: 2/20/13

SPRING 2013

Prof...
CHM333 LECTURES 15: 2/20/13 SPRING 2013 Professor Christine Hrycyna
104
ENZYME KINETICS:
The rate of the reaction catalyzed by enzyme E
A + B P
is defined as
-Δ[A] or -Δ[B] or Δ[P]
Δt Δt Δt
A and B changes are negative because the substrates are disappearing
P change is positive because product is being formed.
Enzyme activity can be assayed in many ways
disappearance of substrate
appearance of product
For example, you could measure
appearance of colored product made from an uncolored substrate
appearance of a UV absorbent product made from a non-UV-absorbent substrate
appearance of radioactive product made from radioactive substrate
Many other ways possible – Just need a way to distinguish the
products from the substrates
The VELOCITY (reaction rate) (product formation of disappearance of substrate/time)
of an enzyme catalyzed reaction is dependent upon the substrate concentration [S].
Velocity related to [S]
The velocity (V) of an enzyme-catalyzed reaction is
dependent upon the substrate concentration [S]
A plot of V vs [S] is often hyperbolic
Michaelis-Menten plot
Graph is not a graph of product formation over time!!!
Enzyme Kinetics: Velocity
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