1997 Ma & Taylor JBC p717, kinetics of monomeric construct

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1997 Ma & Taylor JBC p717, kinetics of monomeric construct Door Mind Map: 1997 Ma & Taylor JBC p717, kinetics of monomeric construct

1. Kinesin type

1.1. Human kinesin construct K332

1.1.1. equivalent to Drosophila 340

1.2. expressed in E. coli, not sure how purified

2. Cited by me in

2.1. 2011 Larry Kinetic Modeling Paper

3. Rates

3.1. MONOMERIC, unbound empty, bind ATP

3.1.1. From Figure 2

3.1.1.1. 9 / micromolar / s in 10 mM NaCl

3.1.1.2. MONOMERIC bound empty binding ATP 500/s maximum?

3.2. MONOMERIC, unbound empty, bind ADP

3.2.1. From Figure 2

3.2.1.1. 4 / micromolar / s

3.3. MONOMERIC bound ATP hydrolysis 200/s

3.3.1. To satisfy 60/s overall rate, they say hydrolysis must be at least 200/s

3.4. MONOMERIC bound ATP, release ATP

3.4.1. probably less than 40/s

3.5. MONOMERIC bound ADP head unbinding 80/s (+/- 10/s) (kdis)

3.5.1. Discussion says 75+/-10 /s

3.5.2. Later in discussion, REACTION 3, head release from ADP state is >300/s

3.6. MONOMERIC bound ADP, release ADP 150 /s or 200 /s

3.6.1. 110 /s from one method, 300/s from another; 150/s from model fitting. A third experiment indicated 400 /s.

3.6.1.1. They seem to say both 150 and 200, but neither with too much confidence in the scheme, since the two methods don't agree

3.6.1.2. In companion paper, they cite the 110/s value only (p. 725, 2nd to last paragraph)

3.7. MONOMERIC bound ADP-P, phosphate release

3.7.1. About 200/s, if using SCHEME 2 and assuming about 200/s for ADP release above.

3.8. Monomeric Vmax 60 /s

3.8.1. hyperbolic fit to rate versus tubulin concentration

4. Assay conditions

4.1. Temperature 20C

4.1.1. Sometimes they say 22C

4.2. Otherwise methods seem to be described in Ma and Taylor 1995 Biochemistry 34 13233 and 13242

4.2.1. No actually have to go back to Sadhu and Taylor JBC 1992

4.2.2. Unsure, but seems to be

4.2.2.1. 25 mM PIPES pH 6.9

4.2.2.2. 5 mM NaCl

4.2.2.3. 2 mM MgCl2

4.2.2.4. 1 mM EGTA

4.2.2.5. sometimes 25 mM KCl in 20 mM Tris pH 7.5

4.2.2.6. Seems to actually be 10 mM NaCl

5. Citation

5.1. Ma, Y. Z., & Taylor, E. W. (1997). Kinetic Mechanism of a Monomeric Kinesin Construct. Journal of Biological Chemistry, 272(2), 717-723. doi:10.1074/jbc.272.2.717

6. Interesting

6.1. The nucleotide-free K332 was more stable than K379 which tends to aggregate with loss of binding activity

7. My assessment

7.1. A lot of these rate constants should be looked at as a reference for zero-force rate constants (i.e. one-head bound) of the dimeric construct. However, there are some caveats

7.1.1. The buffer is not PEM80

7.1.2. Some of their rate constants are derived from models. It is not clear which model is the best, and some of their experiments (e.g. ADP release) have disagreeing results from differing methods

8. companion paper

8.1. Ma & Taylor JBC 1997 p 724