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

Kinesin type

Human kinesin construct K332

equivalent to Drosophila 340

expressed in E. coli, not sure how purified

Cited by me in

2011 Larry Kinetic Modeling Paper

Rates

MONOMERIC, unbound empty, bind ATP

From Figure 2, 9 / micromolar / s in 10 mM NaCl, MONOMERIC bound empty binding ATP 500/s maximum?

MONOMERIC, unbound empty, bind ADP

From Figure 2, 4 / micromolar / s

MONOMERIC bound ATP hydrolysis 200/s

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

MONOMERIC bound ATP, release ATP

probably less than 40/s

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

Discussion says 75+/-10 /s

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

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

110 /s from one method, 300/s from another; 150/s from model fitting. A third experiment indicated 400 /s., They seem to say both 150 and 200, but neither with too much confidence in the scheme, since the two methods don't agree, In companion paper, they cite the 110/s value only (p. 725, 2nd to last paragraph)

MONOMERIC bound ADP-P, phosphate release

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

Monomeric Vmax 60 /s

hyperbolic fit to rate versus tubulin concentration

Assay conditions

Temperature 20C

Sometimes they say 22C

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

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

Unsure, but seems to be, 25 mM PIPES pH 6.9, 5 mM NaCl, 2 mM MgCl2, 1 mM EGTA, sometimes 25 mM KCl in 20 mM Tris pH 7.5, Seems to actually be 10 mM NaCl

Citation

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

Interesting

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

My assessment

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

The buffer is not PEM80

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

companion paper

Ma & Taylor JBC 1997 p 724