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, v o Slope of initial [P]/t at a particular [substrate] is a single point on the Michaelis-Menten graph

4. 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)!

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 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

10. Double displacement (ping – pong) reaction

11. 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

12. Temperature Dependence

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

14. TM1667: glucose isomerase Bandlish et al. Biotech and Bioengineering, 80, 185 – 194 (2002) glucosefructose 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 Appl Microbiol, 97, 1006 – 1014 (2004)

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

22. TM1520: Dihydrodipicolinate reductase

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 – O 2 electrode assay – Radiographic assay – Gel assay