enzyme kinetics
DESCRIPTION
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, v o. - PowerPoint PPT PresentationTRANSCRIPT
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Enzyme Kinetics
Lecture 4CHEM4421 2011
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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.
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Initial Velocity, vo
y = 0.2874x + 0.0251R2 = 0.9994
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4 5
Time (min)
Abs
orba
nce
(410
nm)
Slope of initial [P]/t at a particular [substrate] is a single point on the Michaelis-Menten graph
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Linear (Reciprocal) Plots
The Lineweaver-Burk double-reciprocal plot, depicting extrapolations that allow the determination of the x- and y-intercepts and slope.
Lineweaver-Burk
A Hanes-Woolf plot of [S]/v versus [S], another straight-line rearrangement of the Michalelis-Menten equation.
Hanes-Woolf
You should use Hanes-Woolf, Eadie-Hofstee, or fit the hyperbolic curve (I do not think excel will do this, but Origin will)!
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Demo
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Different Types of Bi-Bi Reactions
Single displacement (sequential)randomordered
Double displacement (ping pong)
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Single displacement Ordered: e.g., lactate dehydrogenase
Random: e.g., creatine kinase
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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.
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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.)
Random, single-displacement bisubstrate mechanism
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Double displacement (ping – pong) reaction
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Double-displacement (ping-pong) bisubstrate mechanisms are characterized by Lineweaver-Burk 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.
Ping-pong bisubstrate mechanism
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Temperature Dependence
V0,max(T)
T (oC)4020 30 50 60 70 80
Arrhenius kinetic behavior
proteindenaturation
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pH dependence
V0
pH
pKa of reaction 1 ~ 4.0
pKa of reaction 2 ~ 9.0
2 124 6 8 10
max
low
Activity decreases due to lysine deprotonationActivity decreases due
to glutamate/aspartate protonation
Maximal activity range
More informative to plot Km and Vmax vs pH, which is most effected?
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TM1667: glucose isomeraseBandlish 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
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TM1155: glucose-6-phosphate dehydrogenase Hansen et al. FEMS Microbiology Letters, 216, 249 – 253 (2002)
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TM1155: glucose-6-phosphate dehydrogenase
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TM0343: 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) sythase
Wu et al., JBC, 278, 27525 – 27531 (2003)
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TM0343: 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase
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TM1469: ATP-dependent glucokinaseHansen and Schönheit. FEMS Microbiology Letters, 226, 405 – 411 (2003)
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TM1193: β-galactosidaseKim et al. J Appl Microbiol, 97, 1006 – 1014 (2004)
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TM1520: Dihydrodipicolinate reductasePearce et al. J. Biochem, 143, 617 – 623 (2008)
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TM1520: Dihydrodipicolinate reductase
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TM0209: ATP-dependent 6-phosphofructokinaseHansen et al. Arch Microbiol, 177, 401 – 409 (2002)
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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