386 Chapter 10. Enzymes and molecular machines[[Student version, January 17, 2003]]
kinesin.
One reasonable-sounding model for the stepping of kinesin might be the following: Suppose
that binding of ATP is a purely chemical step, but its subsequent hydrolysis and release entail
forward motion—apowerstroke.Referring to Figure 10.17b on page 375, this proposal amounts
to assuming that the load force pulls the second or third activation barrier up without affecting
the first one; in the language of Equation 10.16, load reducesk 2 without affectingk 1 ork- 1 .We
already know how such a change will affect the kinetics: Equation 10.19 predicts thatvmaxwill
decrease with load (as observed), whileKMwill also decrease (contrary to observation). Thus the
data in Table 10.1 rule out this model.
Apparently there is another effect of load besides slowing down a combined hydrolysis/motion
step. Schnitzer and coauthors proposed a model almost as simple as the unsuccessful one above
to explain their data. Before describing their proposed mechanism, we must digress to summarize
some prior structural and biochemical studies.
Structural clues The microtubule itself also has a dimeric structure, with two alternating sub-
unit types (Figure 10.21). One of the two subunits, called “β,” has a binding site for kinesin; these
sites are regularly spaced at 8nmintervals. The microtubule has apolarity;the subunits are all
oriented in the same direction relative to each other, giving the whole structure a definite “front”
and “back.” We call the front the “+ end of the microtubule.” Since protein binding is stereospe-
cific (two matching binding sites must be oriented in a particular way), any bound kinesin molecule
will point in a definite direction on the microtubule.
Each head of the kinesin dimer has a binding site for the microtubule, and a second binding site
for a nucleotide, such as ATP. Each kinesin head also has a short (15 amino-acid) chain called the
neck linker.The neck linkers in turn attach to longer chains. The two heads of the kinesin dimer
are joined only via these chains, which intertwine, as shown schematically in Figure 10.21 on page
- As sketched in the figure, the distance between the heads is normally too short for the dimer
to act as a bridge between two binding sites on the microtubule.
One further structural observation holds another clue to the puzzle just mentioned. Chapter 9
metioned that the neck linker adopts strikingly different conformations depending on the occupancy
of the nucleotide-binding site (see Figure 9.11 on page 332). When the site is empty, or occupied
byADP, the neck linker flops between at least two different conformations. When the site contains
ATP,however, the neck linker binds tightly to the core of the kinesin head in a single, well-defined
orientation, pointing toward the “+” end of the microtubule. This allosteric change is critical for
motility: A modified kinesin, with its neck linker permanently attached to the head, was found to
beunable to walk.
Table 10.1:Michaelis–Menten parameters for conventional kinesin stepping at fixed load force. [Data from Schnitzer
et al., 2000.]
load force (pN) vmax(nm s−^1 ) KM(μM)
1.05 813 ± 28 88 ± 7
3.6 715 ± 19 140 ± 6
5.6 404 ± 32 312 ± 49