Figure 1.
The canonical energy landscape for an enzymatic reaction.
is the bound state representing the enzyme - substrate complex;
represents the enzyme - product complex.
Figure 2.
Cartoon of the yeast GK-DNA chimera (in the “open" state) with the DNA spring attached at the sites 60 and 139.
Upon binding of the substrate GMP, the two lobes of the enzyme close on each other. This size conformational motion is a classic example of induced fit. The enzyme structure is PDB 1EX6; the DNA is from the MD simulations of Lankas et al [38], and is kinked in the middle.
Figure 3.
“Low Mg” titration curves (initial reaction speed vs. initial substrate concentration) for the yeast GK chimera in the absence (circles) and presence (triangles) of mechanical stress.
Circles: double-stranded DNA spring with a 4 bp central gap; triangles: double-stranded DNA spring with no gap or nick. Squares: ssDNA spring; this is a confusing control explained in the text. Titrations were performed under the same “low Mg" buffer conditions ( mM KCl and
mM MgCl2) and with the same chimera sample. Here and for Fig. 4, each experimental point represents the average of 4–5 measurements (with the same chimera sample); error bars are the standard deviation. Similar titration curves were also obtained with 3 different (independent constructions of) chimera samples (data not shown), with the same result within experimental error. a) GMP titration for the forward reaction. Enzyme concentration was
nM and initial ATP concentration was
M. b) GDP titration for the reverse reaction. Enzyme concentration was
nM (3 times larger than in (a)) and initial ADP concentration was
M.
Table 1.
The kinetic parameters ,
, and
extracted from the titration data for the forward direction (formation of GDP and ADP) and the reverse reaction (formation of GMP and ATP) in high Mg conditions.
Table 2.
The kinetic parameters ,
, and
extracted from the titration data for the forward and reverse reactions in low Mg conditions.
Figure 4.
“High Mg” titration curves (initial reaction speed vs. initial substrate concentration) for the yeast GK chimera in the absence (circles) and presence (triangles) of mechanical stress.
Circles: double-stranded DNA spring with a 4 bp central gap; triangles: double-stranded DNA spring with no gap or nick. Squares: ssDNA spring. Titrations were performed under "high salt" buffer conditions: mM KCl and
mM MgCl2. a) GMP titration curves for the forward reaction. Conditions were the same as in Fig. 3: enzyme concentration was
nM and and initial ATP concentration was
M. b) GDP titration curves for the reverse reaction. Enzyme concentration was
nM and initial ADP concentration was
M.
Figure 5.
A proposed two-dimensional reaction scheme which can explain the observed effects of mechanical stress.
E, S, and P stand for enzyme, substrates, and products; the upper branch refers to the forward reaction, the lower branch to the reverse reaction. Open and closed refer to the state of the enzyme; is a complex of the enzyme with GMP and ATP, and similarly for the other states.
Table 3.
The coordination states for the reaction substrates and products in both low and high Mg2+ conditions.
Figure 6.
An alternative two-dimensional reaction scheme which is also consistent with the observations.
Here two forms of the enzyme, open and closed, are stabilized by the presence of GDP/ADP and GMP/ATP, respectively. The essentially uni-directional arrows and
can be accomodated within standard transition state theory if one assumes, in both cases, a high barrier for the reverse process. As with the scheme of Fig. 5, we have to assume that under high Mg conditions the arrows in the loop are reversed.