Enzyme Kinetics
Lecture 4
CHEM4421 2011
Michaelis-Menten kinetics model
Seminal work published in 1912 by Leonor
Michaelis (1875–1949) and Maud Leonora
Menten (1879–1960), a German man and
a Canadian woman, cast light on the
reasons why enzymes are so efficient.
Initial Velocity, vo
Absorbance vs. Time (.2 ml enzyme)
Absorbance (410nm)
1.2
1
0.8
0.6
0.4
y = 0.2874x + 0.0251
R2 = 0.9994
0.2
0
0
1
2
3
Time (min)
Slope of initial [P]/t at a
particular [substrate] is a
single point on the
Michaelis-Menten graph
4
5
Linear (Reciprocal) Plots
Lineweaver-Burk
The Lineweaver-Burk double-reciprocal plot,
depicting extrapolations that allow the
determination of the x- and y-intercepts and
slope.
Hanes-Woolf
A Hanes-Woolf plot of [S]/v versus [S],
another straight-line rearrangement of
the Michalelis-Menten equation.
You should use Hanes-Woolf, Eadie-Hofstee, or fit the hyperbolic curve (I do
not think excel will do this, but Origin will)!
Demo
Different Types of Bi-Bi Reactions
Single displacement (sequential)
random
ordered
Double displacement (ping pong)
Single displacement
Ordered: e.g., lactate dehydrogenase
Random: e.g., creatine kinase
Single-displacement bisubstrate mechanism
Single-displacement bisubstrate mechanism. Double-reciprocal plots of the rates observed
with different fixed concentrations of one substrate (B here) are graphed versus a series of
concentrations of A. Note that, in these Lineweaver-Burk plots for single-displacement
bisubstrate mechanisms, the lines intersect to the left of the 1/v axis. Ks are dissocation
constants for A and B as indicated in the superscript.
Random, single-displacement bisubstrate mechanism
Random, single-displacement bisubstrate mechanisms where A does not affect B binding, and
vice versa. Note that the lines intersect at the 1/[A] axis. (If [B] were varied in an experiment
with several fixed concentrations of A, the lines would intersect at the 1/[B] axis in a 1/v versus
1/[B] plot.)
Double displacement (ping – pong) reaction
Ping-pong bisubstrate mechanism
Double-displacement (ping-pong) bisubstrate mechanisms are characterized by LineweaverBurk plots of parallel lines when double-reciprocal plots of the rates observed with different
fixed concentrations of the second substrate, B, are graphed versus a series of
concentrations of A.
Temperature Dependence
pH dependence
M axim al activity
ran ge
pK a of reactio n 2
~ 9.0
pK a o f reaction 1
~ 4.0
m ax
V0
A ctivity decreases d ue
to lysin e deproton ation
A ctivity decreases d ue
to glutam ate/asp artate
p ro to natio n
low
2
4
6
8
10
12
pH
More informative to plot Km and Vmax vs pH, which is most effected?
TM1667: glucose isomerase
Bandlish et al. Biotech and Bioengineering, 80, 185 – 194 (2002)
glucose
fructose
1U of enzyme activity is defined as that catalyzing the formation of 1 μmol product in 1 min
Specific activity is the number of units per mg of protein
TM1155: glucose-6-phosphate dehydrogenase
Hansen et al. FEMS Microbiology Letters, 216, 249 – 253 (2002)
TM1155: glucose-6-phosphate
dehydrogenase
TM0343: 3-deoxy-D-arabino-heptulosonate 7phosphate (DAHP) sythase
Wu et al., JBC, 278, 27525 – 27531 (2003)
TM0343: 3-deoxy-D-arabino-heptulosonate 7phosphate (DAHP) synthase
TM1469: ATP-dependent glucokinase
Hansen and Schönheit. FEMS Microbiology Letters, 226, 405 – 411 (2003)
TM1193: β-galactosidase
Kim et al. J Appl Microbiol, 97, 1006 – 1014 (2004)
TM1520: Dihydrodipicolinate reductase
Pearce et al. J. Biochem, 143, 617 – 623 (2008)
TM1520: Dihydrodipicolinate reductase
TM0209: ATP-dependent 6-phosphofructokinase
Hansen et al. Arch Microbiol, 177, 401 – 409 (2002)
Beyond Photometric Assays
• Many other ways to observe the activity of an
enzyme over time
– Fluorescence (photometric)
– HPLC
– NMR
– O2 electrode assay
– Radiographic assay
– Gel assay