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Enzyme Kinetics

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.

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Enzyme Kinetics

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  1. Enzyme Kinetics Lecture 4 CHEM4421 2011

  2. 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.

  3. Initial Velocity, vo Slope of initial [P]/t at a particular [substrate] is a single point on the Michaelis-Menten graph

  4. Linear (Reciprocal) Plots Lineweaver-Burk Hanes-Woolf The Lineweaver-Burk double-reciprocal plot, depicting extrapolations that allow the determination of the x- and y-intercepts and slope. 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)!

  5. Demo

  6. Different Types of Bi-Bi Reactions Single displacement (sequential) random ordered Double displacement(ping pong)

  7. Single displacement Ordered: e.g., lactate dehydrogenase Random: e.g., creatine kinase

  8. 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.

  9. 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.)

  10. Double displacement (ping – pong) reaction

  11. Ping-pong bisubstrate mechanism 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.

  12. Temperature Dependence

  13. pH dependence More informative to plot Km and Vmaxvs pH, which is most effected?

  14. 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

  15. TM1155: glucose-6-phosphate dehydrogenase Hansen et al. FEMS Microbiology Letters, 216, 249 – 253 (2002)

  16. TM1155: glucose-6-phosphate dehydrogenase

  17. TM0343: 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) sythase Wu et al., JBC, 278, 27525 – 27531 (2003)

  18. TM0343: 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase

  19. TM1469: ATP-dependent glucokinase Hansen and Schönheit. FEMS Microbiology Letters, 226, 405 – 411 (2003)

  20. TM1193: β-galactosidase Kim et al. J ApplMicrobiol, 97, 1006 – 1014 (2004)

  21. TM1520: Dihydrodipicolinatereductase Pearce et al. J. Biochem, 143, 617 – 623 (2008)

  22. TM1520: Dihydrodipicolinatereductase

  23. TM0209: ATP-dependent 6-phosphofructokinase Hansen et al. Arch Microbiol, 177, 401 – 409 (2002)

  24. 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

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