Supplementary Material
Determination of the microscopic rate constants:
Basal GTP binding and hydrolysis (K1, k2 and K1’, k2’). The chemical step is
rate-limiting for the basal GTPase reaction of cpSRP54, because the maximal rate
constant of GTP hydrolysis (0.017 min-1; Figure 2A) is 4  104 -fold slower than the rate
at which GTP dissociates from the enzyme active site (10.4 s-1; Figure 3E). Therefore,
the K1/2 value obtained from the data in Figure 2A is equal to K1, the equilibrium
dissociation constant for GTP, and the kmax value from the same figure is equal to k2, the
rate constant for GTP hydrolysis from the GTP•cpSRP54 complex. For the same reason,
the chemical step is rate-limiting for the basal GTPase reaction of cpFtsY. Therefore, the
K1/2 value obtained from the data in Figure 2B is equal to K1’, the dissociation constant
for GTP, and the kmax value obtained from the same figure is equal to k2’, the rate
constant for GTP hydrolysis from the cpFtsY•GTP complex. The values of K1 and K1’
were also determined independently by fluorescence assays (Figure 3C) as described in
the text.
GDP binding to cpSRP54 and cpFtsY (K3 and K3’). The binding affinities of
GDP for both proteins were determined by using GDP as a competitive inhibitor of the
basal GTPase reaction, as described previously (Peluso et al., 2000), and by fluorescence
assays (Figure 3D) as described in the text.
Nucleotide dissociation rate constants (k-1, k-1’ and k-3, k-3’). The rate constants
for nucleotide dissociation from each protein were measured using fluorescent mant-GTP
and mant-GDP in pulse-chase experiments (Figure 3E and 3F and data not shown) as
described in the text.
Nucleotide association rate constants (k1, k1’ and k3, k3’). The rate constants for
binding of GTP and GDP to both proteins were obtained from the equilibrium
dissociation constant and the dissociation rate constant for each nucleotide, determined as
described above, using kon = koff / Kd.
Rate constant for complex formation (k4). The association rate constant between
cpSRP54 and cpFtsY was not determined directly due to the lack of a direct proteinprotein binding assay, and was estimated from the value of kcat/Km for the stimulated
GTPase reaction; this value provides a lower limit for k4, as explained in the Results.
Rate constant for GTP hydrolysis in the GTP•cpSRP54•cpFtsY•GTP complex (k5).
This rate constant was derived from the value of kcat determined from the stimulated
GTPase reaction between cpSRP54 and cpFtsY (Figure 4, circles). Several observations
suggest that product release is not rate-limiting for kcat. First, the value of kcat is the
same, within experimental error, as the rate constant of GTP hydrolysis from the
GTP•
cpSRP54•cpFtsY•GTP complex determined under single turnover conditions (data not
shown). Second, the time course for the reaction: GTP•cpSRP54•cpFtsY•GTP  products
is consistent with a single exponential rate without exhibiting a burst phase
(Supplementary Figure 1). Thus, steps prior to GTP hydrolysis, rather than product
release, is rate-limiting for the stimulated GTPase reaction from the
GTP•
cpSRP54•cpFtsY•GTP complex. Therefore, kcat represents the sum of rate constants
for hydrolysis of the two GTPs from the GTP•cpSRP54•cpFtsY•GTP complex (k5) and may
be limited by either the chemical step itself, or a conformational change prior to GTP
hydrolysis.
Rate constant for complex dissociation (k6). For the same reasons stated in the
previous paragraph, the value of kcat sets a lower limit for the rate of product release (k6),
which has not been directly measured in this study.
Supplementary Figure 1 The time course for GTP hydrolysis from the cpSRP54•cpFtsY
complex shows no obvious burst phase. The reaction was carried out in the presence of
12.5 M cpSRP54, 15 M cpFtsY, and 100 M GTP doped with trace amounts of GTP*;
the high concentration of protein relative to GTP is used to maximize the chance of
observing the presence of a burst phase. The different symbols represent data from two
independent measurements. The line is a fit of the initial part of the time course to a
single-exponential rate equation.
Supplementary Figure 2 cpSRP43 has no significant effect on the stimulated GTPase
reaction of cpSRP54 and cpFtsY. (A) Rates of the stimulated GTPase reactions were
determined for cpSRP54 (100 nM) and increasing concentration of cpFtsY in the
presence of 100 M GTP, with () and without () 1 M cpSRP43 present. Fits of the
data to eq 2 in the Methods gave kcat values of 49.5 and 45.5 min-1 and Km values of 1.7
and 2.3 M in the presence and absence of cpSRP43, respectively. (B) The rate of the
stimulated GTPase reaction: GTP•cpSRP54 + cpFtsY•GTP  2GDP + Pi does not change
significantly over a range of cpSRP43 concentrations. Note that the Kd for formation of
the cpSRP54•cpSRP43 complex is in the low nanomolar range (Hermkes et al., 2006), so
a stable cpSRP54•cpSRP43 complex is formed at lowest cpSRP43 concentration used in
this experiment.
References
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Peluso, P., Herschlag, D., Nock, S., Freymann, D. M., Johnson, A. E., and Walter, P.
2000. Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its
receptor. Science 288: 1640-1643.
Supplementary Figure 1
Supplementary Figure 2