From the
The kinetic behavior of a H
The ES leak and the EH
leak mutants provide a mechanism for substrate-induced and
substrate-inhibited proton leakage, respectively. Furthermore,
substrate efflux down a concentration gradient is inhibited by a
membrane potential (inside negative) under uncoupled conditions in the
case of an ES leak but not in the case of an EH leak.
The properties
of the mutants mimic those of various transport mutants that have been
described, in particular mutants of the lactose transport protein of
Escherichia coli. The analysis offers general means for
targeted experimentation, which allows discrimination between various
types of transport mutants.
It is generally believed that catalysis by a proton symporter
proceeds via the formation of a ternary complex among substrate,
proton, and carrier as depicted in Fig. SIA. The binding
sites for proton and substrate are oriented simultaneously toward
either the outside or the inside of the cell. The
``unloaded'' states of the carrier, E
The kinetic behavior of the wild-type enzyme and the
uncoupled mutants was analyzed by numerical solution of the steady
state equations using the computer program CACES (Lolkema, 1993). The
basic set of rate constants pertinent to Fig. SIA used
in the simulations was as follows. The affinity constant for the
substrate was 1 mM with rate constants of 1000
mM
A membrane potential
Cycle Ia
represents facilitated diffusion of the substrate catalyzed by the
unprotonated carrier. It prevails at high pH values and compensates for
the decreased rate of uptake at higher pH values observed with the
wild-type enzyme. At low pH values, the major pathway proceeds via the
translocation of the ternary complex. Cycle Ib depicts a transport mode
for the proton catalyzed by the enzyme-substrate complex
E
Introduction of the EH leak results in two similar cycles. Cycle IIa
provides a pathway for the proton, resulting in a proton leak in the
absence of substrate. Cycle IIb represents a facilitated diffusion
cycle for the substrate catalyzed by the protonated carrier. It
compensates for the decrease in the rate at low pH values observed with
the wild-type enzyme (Fig. 1, A and B,
The affinity constants for the substrate of
the transport activities shown in Fig. 1show a sigmoidal
increase with pH. Introduction of the leaks does not result in dramatic
changes in the K
Both in the ES leak and
the EH leak the carrier shifts from symport to uniport and vice
versa as a function of pH. The different cycles introduced by the
two leaks result in significant different kinetic behavior of the
enzyme under various conditions (see below).
On-line formulae not verified for accuracy
In the case of a mutant with an ES leak, the
ratio of the initial rates of uptake of proton and substrate decreases
sigmoidally from 1 at low to 0 at high pH values both with
In the case
of an ES leak, both the stoichiometries from initial rates and
accumulation are higher in the presence of a membrane potential than in
the presence of a pH gradient of the same magnitude. This is just
opposite for the EH leak. The effects of the membrane potential and pH
gradient on the accumulation of substrate are analyzed in more detail
in Fig. 3. The difference in accumulation in response to a
membrane potential or a pH gradient increases with increasing magnitude
of the forces. Analysis of the degree of coupling, n
The pH profiles of the efflux activities
of the wild-type enzyme and the mutants with the ES leak and the EH
leak are similar to the profiles shown in Fig. 1for influx.
Mutants with the ES leak are characterized by a high rate of efflux at
high pH, which is catalyzed by the unprotonated carrier as described by
cycle Ia. Both translocation steps in cycle Ia are affected by the
membrane potential. The membrane potential tends to speed up efflux
through its effect on the transition between E
Functional analysis of mutant secondary transporter enzymes
may yield important information about residues involved in substrate
and proton binding, translocation, coupling, etc. Such studies will
also improve our knowledge about the kinetic mechanism of the wild-type
enzyme. However, in spite of major efforts by a number of laboratories
it has proven to be difficult to associate (mutated) residues with
specific steps in the kinetic mechanism. In part, this is caused by the
complicated relation between the kinetic behavior and the role of
individual residues. In this study we analyze the kinetic behavior of
two types of uncoupled mutants, those with mobile enzyme-substrate (ES
leak) and enzyme-proton (EH leak) complexes. The two mechanisms of
uncoupling are directly linked to the binding of substrate and proton
(see Introduction), and, therefore, mutated residues that result in
either type of uncoupling are involved in the events that follow upon
the binding of substrate or proton to the carrier. The ability to
discriminate between residues involved in substrate or proton binding
adds a lot of detail to the role of the individual residues. It should
be stressed that the analysis is general to symporters, irrespective of
transported cation and/or substrate.
The aim of the present study is
to describe the steady state kinetic characteristics of the ES leak and
EH leak type of mutants by simulating those experiments that are
particularly discriminative. The major conclusions are as follows: (i)
the two types of mutants can be discriminated easily by various
criteria (summarized in ); (ii) in partially coupled
systems, the degrees of coupling inferred from initial rates and
accumulation are not the same; and (iii) the coupling characteristics
are largely independent of the rate constants in the kinetic scheme.
Numerical analyses as used in this study require the assignment of
numerical values to the rate constants pertinent to the kinetic scheme.
The basic set of rate constants we have used represents a rather simple
enzyme that is symmetrical with respect to the two sides of the
membrane and in which the binding equilibria are rapid relative to the
translocation equilibria. This choice improves the comprehensibility of
the results. However, this set may not always be valid, e.g. functional asymmetry of secondary transporters has frequently been
observed, and the binding equilibria are not necessarily fast relative
to the translocation equilibria (Viitanen et al., 1983; Page,
1987; Loo et al., 1993). For this reason we have tested
various additional schemes in which non-rapid binding equilibria,
asymmetry, and cooperativity between the binding sites were introduced.
The outcome of these analyses is that the coupling characteristics of
the mutants described in this study do not differ much between the
various schemes even though other kinetic parameters may be very
different. Thus, the phenomena in the presented plots represent trends
that are largely independent of the assumptions made in the original
model. Importantly, the numbers should not be compared literally with
any experimental data. Also, the analysis given in this study is one of
extreme cases; the mutant carriers are characterized by one mobile and
one immobile binary complex. Real mutations may affect the mobility of
the two binary complexes differently, i.e. the
E
The lactose transport protein of E. coli is the most extensively studied bacterial transport system (for
reviews see Kaback (1990); Poolman and Konings(1993)), and reports on
LacY mutant characteristics provide ample evidence for the existence of
ES leak and EH leak types of mutants (). Various mutants of
LacY have been isolated that are defective in uphill transport of
galactosides but have a more or less normal
H
It is
tempting to classify the various mutations of as ES leak
type or EH leak type. However, in general, the experimental data is too
incomplete to assign the mutants unambiguously to a particular leak
type. The present analysis offers relatively simple means for further
experimentation which should give a more solid basis for the
classification. The data in should be regarded as evidence
for the validity of the two mechanisms of uncoupling analyzed in this
paper.
We thank Prof. Dr. W. N. Konings for critical reading
of the manuscript and many helpful suggestions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-substrate symporter
has been studied in which in addition to the unloaded (E) and
fully loaded states (E
S
H) of the carrier also one
of the binary complexes (E
S or E
H) may
reorient its binding sites. This results in two types of uncoupled
mutants, the ES leak and the EH leak type. The effects of pH and
substrate concentration (pS) on the coupling of transport have been
analyzed. In the enzyme with the ES leak, the proton:substrate
stoichiometry (v(H
)/v(S)) and the
substrate accumulation levels decrease sigmoidally from fully coupled
at low pH to completely uncoupled at high pH. Importantly, the coupling
inferred from initial rate measurements is higher than from steady
state accumulation levels. In the enzyme with the EH leak, the coupling
inferred from the accumulation levels increases from no coupling at low
pH to full coupling at high pH and saturating substrate concentration.
The v(H
)/v(S) increases sigmoidally
with pH from <1 to >1 and is highly dependent on pS. At each pH
value a substrate concentration can be found that results in apparent
complete coupling between the two fluxes.
and E
, and the fully loaded states,
E
S
H and
E
S
H, are mobile forms of the carrier,
i.e. they can reorient the binding sites spontaneously. In
contrast, the binary complexes between carrier and either substrate are
immobile. The membrane potential is believed to exert its kinetic
effect on one of the mobile forms, in the case of LacY, the lactose
transport protein of E. coli, the unloaded carrier (Garcia
et al., 1983). The coupling of substrate and proton flux is a
direct consequence of the mobile and immobile states of the carrier
(for a recent discussion see Krupka(1993)). Reorientation of the
binding sites and, therefore, transport takes place only after both
proton and substrate have bound to their respective binding sites.
Binding of one of the two substrates to the unloaded carrier changes
the carrier from a mobile to an immobile state. The carrier becomes
``locked'' in a state with the binding sites oriented toward
the side of the membrane from which the binding took place. Both
substrates can lock the carrier independently of each other in the
immobile state. Therefore, we postulate that two types of mutants with
an uncoupled phenotype can be found: the ``ES leak'' type and
the ``EH leak'' type (Fig. SI, B and
C, respectively). Binding of the substrate to the unloaded
carrier in the ES leak type does not result in locking of the carrier,
whereas binding of the proton does. In the EH leak type this is just
the other way around.
Figure SI:
Scheme IKinetic schemes for the wild-type
proton-symporter (A), the ES leak type of mutant (B),
and the EH leak type of mutant (C). Subscripts o and i denote
the external and internal orientation of the binding sites on the
enzyme, respectively. The steps that are affected by the membrane
potential are indicated by .
In the present paper we analyze the behavior
of the ES leak and the EH leak types of uncoupled mutants in steady
state kinetic measurements. It is demonstrated that the degree of
coupling may be very different when inferred from initial rate
measurements or steady state accumulation levels. The analysis suggests
ways to discriminate relatively easily between mutants with an ES or EH
leak. This pinpoints the mutated residues to the interactions between
carrier and substrate or carrier and proton. Some properties of the
hypothetical enzymes are strikingly similar to those of various LacY
mutants listed in .
s
and 1000
s
, respectively. The pK of the proton binding site
was 7 with 1000 µM
s
and 100 s
for the rate constants. The rate
constants pertinent to the translocation of the unloaded carrier and
the ternary complex were 20 s
in both directions.
This set corresponds to a symmetrical carrier with rapid binding
equilibria relative to the translocation equilibria. In a second set
the binding equilibria were made slow relative to the translocation
equilibria by increasing the rate constants of the latter to 2000
s
. This set will be referred to as non-rapid binding
equilibria. The rate constants for the translocation of the binary
complexes were set to zero in the kinetic scheme representing the
wild-type carrier. Unless otherwise stated, in the ES leak type of
mutant (Fig. SIB) the rate constants for the
translocation of the enzyme-substrate complex E
S were made
identical to the translocation rate constants of the wild-type
translocations. The same adjustment was made to the rate constants for
the translocation of the protonated carrier E
H in the EH leak
type of mutant (Fig. SIC).
was included in the simulations by affecting the forward
and backward rate constants (k
and
k
, respectively) of selected translocation
equilibria as follows: k
=
k
exp(0.5
/RT) and
k
=
k
exp(-0.5
/RT).
Kinetics of the Wild-type Enzyme and Uncoupled
Mutants
The maximal rate of proton motive force
(pmf)(
)
-driven substrate uptake catalyzed by the
fully coupled enzyme follows a bell-shaped curve when simulated at
different pH values, irrespective of whether the binding steps are fast
or slow relative to the translocations (Fig. 1, A and
B, asterisk). The pH dependence is the result of two
counteracting steps in the catalytic cycle, i.e. the
protonation and deprotonation of the carrier at the external and
internal face of the membrane, respectively (see
Fig. SIA). Since both steps are obligatory for turnover,
the highest rates are observed at intermediate pH values. At lower pH
values the enzyme piles up in state E
H (state
6), at higher pH the enzyme piles up in state E
:S
(3). The highest maximal rate is observed at pH 7 (the pK of
the proton binding sites) in the case of facilitated influx or efflux
(data not shown) and is shifted toward more acidic values in the
presence of a pmf (Fig. 1).
Figure 1:
The maximal rate of substrate uptake of
the wild-type enzyme (*), the ES leak mutant (), and the EH leak
mutant (
) at different pH values. A, rapid binding
equilibria. B, non-rapid binding equilibria. The rate
constants used in the simulations are described under ``Materials
and Methods.'' The initial rates were calculated in the presence
of a pH gradient, inside alkaline, of 1 unit (pmf = -60
mV). The indicated pH value is that of the external
pH.
In the case of the ES leak
(Fig. SIB), the maximal rate of uptake increases with pH
and reaches a plateau at alkaline pH values (Fig. 1, A and B, ). The ES leak opens up two alternative
routes for turnover of the carrier (cycles Ia and Ib).
S. Under conditions of accumulation of the substrate
inside the cell, i.e. when state E
S
becomes populated, the cycle provides a slip for the proton.
).
At low pH values cycle IIb is the major pathway, whereas at high pH the
major pathway is via states E
and
E
.
values. At the pH
optimum the affinity constants range from 0.6 to 1.1 mM and
from 0.5 to 1.7 mM in the case of rapid binding equilibria and
non-rapid binding equilibria, respectively.
Proton:Substrate Stoichiometries
The coupling
stoichiometry of secondary transporters is inferred either from the
accumulation of the substrate inside the cells in the presence of a pmf
of known magnitude or from the ratio of the initial rates of uptake of
the substrate and the proton. Thermodynamic considerations of the
catalyzed symport reaction require that at equilibrium the force on the
substrate is counteracted by the force on the proton.
and
pH as driving force (Fig. 2A). At low pH, the
major pathway is the same as in the wild-type enzyme and, consequently,
the two fluxes are fully coupled. Increasing the pH results in an
increasing contribution of cycle Ia to the flux with a subsequent
decrease in the rate of proton influx. The stoichiometries are largely
independent of the substrate concentration when the binding equilibria
are fast (Fig. 2A, inset). Increasing the rate
constants for the translocations has no effect on the coupling at low
substrate concentrations but results in reduced coupling at higher
substrate concentrations (not shown). The accumulation levels under
zero flux conditions for the substrate show a similar dependence on the
pH. Accumulation is high at low pH values and negligible at high pH
values (Fig. 2B). Also, the accumulation levels are
independent of the substrate concentration in the case of rapid binding
equilibria (Fig. 2B, inset) and are reduced in
the case of non-rapid binding equilibria at high substrate
concentrations. An important difference between the two experimental
approaches is that at a given pH value the coupling inferred from
accumulation ratios is significantly lower than from initial rate
measurements.
Figure 2:
The
coupling stoichiometry as a function of pH for the ES leak (A,
B) and EH leak (C, D) mutants. PanelsA and C show the ratio of the initial rates of
uptake of proton and substrate. PanelsB and D show the accumulation of the substrate in the cell under
conditions of zero flux for the substrate (kinetic steady state). The
rates and accumulation levels were calculated in the presence of a pH
gradient () and a membrane potential (
) of -60 mV. In
the case of a fully coupled enzyme this would result in an accumulation
ratio of 10. The insets show the dependences on the substrate
concentration in the presence of a pH gradient of -60 mV at pH
5.5 (▾), pH 7 (
), and pH 8.5 (
). The intermediate pH
in the inset of Fig. 2C is pH 6 (
). The y axis in the inset of Fig. 2C is
logarithmic.
The pH profile of the mutant with an EH leak is
opposite to that of the mutant with an ES leak. The ratio of the
initial rates of proton uptake and substrate uptake increases with
increasing pH (Fig. 2C). The ratio ranges from values
below 1 at low pH to values above 1 at high pH, showing that the proton
flux can be slower or faster than the substrate flux depending on the
conditions. In contrast to what was observed with the ES leak, already
with the rapid binding equilibria, the ratio of proton to substrate
flux is strongly dependent on the substrate concentration
(Fig. 2C, inset). With the non-rapid binding
equilibria the curves in the inset of
Fig. 2C shift to the right, resulting in a lower proton
to substrate flux ratio at a fixed substrate concentration. Increasing
concentrations of substrate decrease the degree of coupling from
initial rates by pulling the enzyme in the ternary complex, thereby
reducing the uncoupled proton flux via cycle IIA. The EH leak results
in accumulation levels that also increase sigmoidally with pH
(Fig. 2D). The substrate flux follows the uncoupled
pathway via cycle IIb at low pH and the coupled pathway of the
wild-type enzyme at high pH. The uncoupled pathway does not result in
accumulation, independent of the substrate concentration used. Toward
higher pH values the accumulation becomes more and more dependent on
the substrate concentration, resulting in full coupling at high
substrate concentrations (Fig. 2D, inset). In
the case of non-rapid binding equilibria the concentration of substrate
needed to achieve full coupling at high pH is higher (data not shown).
The effect of the substrate concentration on the degree of coupling is
opposite for initial rate measurements and accumulation.
(see Equation 1), indicates that in both types of mutants the
coupling does not change with the membrane potential but decreases with
the pH gradient in the ES leak and increases with the pH gradient in
the EH leak (Fig. 3, insets). A change in the degree of
coupling requires a change in the relative contributions of coupled and
uncoupled pathways. With the ES leak, the relative contributions of the
uncoupled pathway via cycle Ia and the coupled pathway are determined
by the state of protonation of the carrier on the outside. Therefore,
increasing the pH gradient by increasing the internal pH will not
result in higher accumulation levels, i.e. the degree of
coupling decreases. With the EH leak, the increase of the internal pH
favors deprotonation of the carrier on the inside, which results in an
increase of the coupled relative to the uncoupled pathway (cycle IIb),
i.e. the degree of coupling increases.
Figure 3:
Accumulation efficiency in response to a
membrane potential and a pH gradient. The accumulation under zero flux
condition for the substrate was calculated in the presence of an
external substrate concentration of 1 mM and at pH 7. The pmf
consisted either of a membrane potential () or a pH gradient
(
). The inset shows the degree of coupling,
n
(Equation 2).
The Proton Leak under Zero Flux Conditions for the
Substrate
The wild-type proton symporter does not catalyze net
proton transport after equilibration of the substrate inside the cell.
A mutant with an ES leak cannot transport protons in the absence of
substrate. However, the rate of proton influx under zero flux
conditions for the substrate increases rapidly with increasing external
substrate concentrations (Fig. 4, ). Apparently, the
substrate induces a H
leak corresponding to cycle Ib
in the kinetic scheme for the mutant with the ES leak. The proton
symporter with the EH leak confronts the cell with a continuous influx
of protons via cycle IIa. Fig. 4(
) shows the inhibition of
the proton flux by equilibration of the cells with substrate. The
presence of substrate at both sides of the membrane reduces the flux
through cycle IIa by pulling the enzyme in the enzyme-substrate complex
(E
S
H).
Figure 4:
Substrate-induced and -inhibited proton
leaks. The net proton flux was calculated under condition of zero
substrate flux, i.e. the substrate is equilibrated over the
membrane, and in the presence of a pH gradient of -60 mV. ,
ES leak. The rate constants for the translocation of the ternary
E
S
H and binary E
S complex were set to 200
s
. The association and dissociation rate constants
for the binding equilibrium between the proton and the
E
S complex were set to 5000
µM
s
and 500
s
. The external and internal pH values were 8 and 9,
respectively. Under these conditions the substrate accumulation ranged
from 1.49 at 0.5 mM to 1.28 at 10 mM of substrate.
, EH leak. The rate constants for the translocation of the
unloaded carrier and binary E:H complex were set to 200
s
. The external and internal pH were 6 and 7,
respectively. The accumulation of the substrate ranged from 1.44 at 0.5
mM to 1.73 at 10 mM
substrate.
Kinetic Effect of the Membrane Potential under Uncoupled
Conditions
Thermodynamically, the pH gradient and the membrane
potential are equivalent, i.e. a pH gradient and a membrane
potential of the same magnitude result in the same level of
accumulation of the substrate by the wild-type enzyme. Kinetically, the
two gradients are not equivalent since they act on different steps in
the kinetic scheme. While the pH gradient reflects different external
and internal proton concentrations, the membrane potential affects the
rate constants for the isomerization of the unloaded carrier
(Fig. SI). A membrane potential of physiological polarity speeds
up the transition from state E to E
and inhibits the transition in the opposite direction. Therefore,
the rate of efflux of substrate down a concentration gradient catalyzed
by a wild-type symporter is inhibited by a membrane potential of normal
polarity (inside negative).
S
and E
S
but inhibits efflux through its
effect on the transition between E
and
E
. The latter step becomes rate-limiting as the
membrane potential increases. The net effect is an inhibition of the
rate of efflux by the membrane potential under conditions where the
carrier catalyzes essentially uniport of the substrate (Fig. 5,
). Mutants with an EH leak show high rates of efflux at low pH
values. Cycle IIb is responsible for the efflux. None of the steps in
this cycle are affected by the membrane potential, and, consequently,
the rate of efflux under these conditions is not significantly affected
by the membrane potential (Fig. 5,
).
Figure 5:
Inhibition of efflux by the membrane
potential under uncoupled conditions. The rate of efflux was calculated
at pH 8 for the ES leak () and at pH 6 for the EH leak (
).
The internal substrate concentration was 5 mM. At the same pH
values, the substrate accumulation ratios of substrate at a membrane
potential of -120 mV and 1 mM of external substrate are
1.85 and 1.9 in the case of the ES leak and EH leak, respectively. The
wild-type enzyme would accumulate the substrate by a factor of 100 at a
membrane potential of -120 mV.
S complex becomes more mobile than the
E
H complex or the other way around. The observed
phenotype will be according to the binary complex with the highest
mobility, but the experimental behavior may differ in details from the
analysis given here.
:galactoside stoichiometry. This apparent discrepancy
in the results is also observed in the mutants with the ES and the EH
leak. It follows that n
n
(Equations 1 and 2). In the case of the ES leak this is caused by
the presence of substrate inside the cell under conditions of
accumulation. A significant internal substrate concentration introduces
a futile cycle for the substrate via cycle Ib. Substrate that enters
the cell via the ternary complex leaves the cell again via the ES leak
while the proton stays inside. In the case of the EH leak the proton
flux proceeds via the EH leak (cycle IIa) in the absence of substrate.
Addition of the substrate induces a flux via the ternary complex,
thereby decreasing the flux through the proton leak (see
Fig. SIC). At a particular substrate concentration the
flux through the EH leak changes its direction from out-to-in to
in-to-out. This is the point where the net substrate flux starts
exceeding the net proton flux. Both substrate and proton enter the cell
solely via the ternary complex, but part of the protons leave the cell
again via the leak. The concentration needed to invert the flux through
the EH leak depends strongly on the pH under the rapid binding
equilibrium assumption (Fig. 2C, inset).
Nevertheless, at each pH value a substrate concentration exists at
which the coupling stoichiometry from initial rate measurements equals
1 (Fig. 6). Thus, a mutant with an EH leak may demonstrate an
apparent full coupling between the proton and the substrate flux but,
nevertheless, be unable to accumulate the substrate. The double mutant
A177V/H322N of LacY is a clear example of this phenotype
().
Figure 6:
Apparent complete coupling by the EH leak
mutant from initial rate measurements. The substrate concentration at
which the initial rates of uptake of proton and substrate are equal is
plotted as a function of pH. The driving force consisted of a pH
gradient of -60 mV. The indicated pH is the external one.
Substrate concentrations above and under the line result in proton:substrate stoichiometries that are lower and
higher than 1, respectively. , rapid binding equilibrium;
,
non-rapid binding equilibrium.
A wild-type proton symporter functioning at full
activity will not significantly decrease the steady state proton motive
force. The primary proton pumps can easily compensate for the
substrate-coupled influx of protons via the carrier. This may be
different when cells overproduce a transporter. Then, the increased
``load'' on the pmf may result in a decreased steady state
value of the pmf as is observed after addition of lactose to E.
coli cells overexpressing the LacY protein (Brooker, 1991; King
and Wilson, 1990b). On the other hand, overexpression of some LacY
mutants (A177V/A177T, A177V/K319N, Y236F/Y236H/Y236N/Y236S, see
) results in a lowering of the pmf even in the absence of
substrate. In the LacY-A177V mutant, this defect is reduced in the
presence of the galactoside TDG, which restores the pmf to near normal
values (King and Wilson, 1990b). The properties of this mutant are
consistent with an EH leak (Fig. 4). The presence of substrate at
both sides of the membrane reduces the flux through cycle IIa by
pulling the enzyme in the enzyme-substrate complex. The double mutant
A177V/K319N also exhibits a proton leak, which is reflected in a
lowered pmf and an increased H leakage, but, in
contrast to the single A177V mutant, this leak is enhanced in the
presence of the non-metabolizable substrate TDG (Brooker, 1991).
Expression of mutant LacY proteins in which Arg-302 is substituted for
Ser, His, or Leu also causes a sugar-dependent H
leak
(and lowering of
pH) (Matzke et al., 1992). The behavior
of all these mutants mimics that of transport proteins with an ES leak
pathway (Fig. 4). The enzyme-substrate complex functions as a
facilitator for proton transport (cycle Ib), which is driven by the
pmf. The presence of substrate at both sides of the membrane pulls the
enzyme in the substrate-associated state. Saturation of the carrier
with substrate results in the highest proton leak activity.
Table: 0p4in
King and Wilson, 1990a.(119)
Table:
The kinetic behavior of mutant
enzymes with an ES leak and an EH leak
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.