10. In this example, using the same data range for linear regression
analysis for all ATP concentrations works well. However, sometimes it is necessary to use different ranges at high and low
concentration. For example, if there is larger noise on the data
or some artifacts at the beginning of the reaction like a lag, it
can be better to fit the low concentration data over a longer
time range. The MARS analysis software also has a “maximum
of slope” function, which can be used to find the initial rates. In
this case, it is important to choose the right width for slope
calculation (at least 10 data points, better 20 or more).
11. P i binding to MDCC-PBP occurs in two steps: initial, loose
binding, followed by a conformation change that closes the P i
binding cleft [5]. The kinetics of P i binding show a hyperbolic
dependence of the observed rate constant of binding on the
concentration of the excess compound (P i or MDCC-PBP).
The rate constant of cleft closure (reached at high concentrations) will therefore pose an upper limit on the rate constant of
P i release that can be measured. At 5
C the dissociation
constant for the initial loose binding step is 4.5 μM and the
rate constants for closing and opening are 317 s
À1 and 4.5 s
À1 ,
respectively [5]. At 20
C it is more difficult to resolve the rate
constant for cleft closure, as it is reaching the limit of time
resolution of stopped-flow instruments, but more than
1000 s
À1 has been measured at concentrations above 50 μM
(Martin Webb, personal communication).
12. If the rate of P i binding to PBP is tenfold higher than the rate
constant of the reaction under study, the error in the measured
rate constant is less than 10%, even if the short lag phase due to
the P i binding rate is ignored in the data fitting. However, P i
release rates only two-threefold slower than P i binding can also
be measured, if data are analyzed more carefully with kinetic
modeling software, including the rate of P i binding to PBP.
13. It is ideal to have the cleavage (step 2) and/or P i release (step
3) kinetically isolated from the binding since the traces can be
analyzed by single- or multi-exponential curve fitting to extract
the rate constants for these steps. In practice, it is often difficult
to reach high enough enzyme concentrations for a clear separation of the binding step. In this case, the true rate constants
will be underestimated in simple-exponential fits. For more
accurate rate constants, data can be analyzed using kinetic
simulation software, e.g., Dynafit or KinTek Explorer.
14. To simplify the mechanism, we assume here that chemical
cleavage and P i release are both irreversible. Regarding P i
release, this is the case for most enzymes, since P i affinity is
generally very low. In addition, free P i is trapped by the binding
to MDCC-PBP making the reaction quasi-irreversible. In
314
Simone Kunzelmann
analysis for all ATP concentrations works well. However, sometimes it is necessary to use different ranges at high and low
concentration. For example, if there is larger noise on the data
or some artifacts at the beginning of the reaction like a lag, it
can be better to fit the low concentration data over a longer
time range. The MARS analysis software also has a “maximum
of slope” function, which can be used to find the initial rates. In
this case, it is important to choose the right width for slope
calculation (at least 10 data points, better 20 or more).
11. P i binding to MDCC-PBP occurs in two steps: initial, loose
binding, followed by a conformation change that closes the P i
binding cleft [5]. The kinetics of P i binding show a hyperbolic
dependence of the observed rate constant of binding on the
concentration of the excess compound (P i or MDCC-PBP).
The rate constant of cleft closure (reached at high concentrations) will therefore pose an upper limit on the rate constant of
P i release that can be measured. At 5
C the dissociation
constant for the initial loose binding step is 4.5 μM and the
rate constants for closing and opening are 317 s
À1 and 4.5 s
À1 ,
respectively [5]. At 20
C it is more difficult to resolve the rate
constant for cleft closure, as it is reaching the limit of time
resolution of stopped-flow instruments, but more than
1000 s
À1 has been measured at concentrations above 50 μM
(Martin Webb, personal communication).
12. If the rate of P i binding to PBP is tenfold higher than the rate
constant of the reaction under study, the error in the measured
rate constant is less than 10%, even if the short lag phase due to
the P i binding rate is ignored in the data fitting. However, P i
release rates only two-threefold slower than P i binding can also
be measured, if data are analyzed more carefully with kinetic
modeling software, including the rate of P i binding to PBP.
13. It is ideal to have the cleavage (step 2) and/or P i release (step
3) kinetically isolated from the binding since the traces can be
analyzed by single- or multi-exponential curve fitting to extract
the rate constants for these steps. In practice, it is often difficult
to reach high enough enzyme concentrations for a clear separation of the binding step. In this case, the true rate constants
will be underestimated in simple-exponential fits. For more
accurate rate constants, data can be analyzed using kinetic
simulation software, e.g., Dynafit or KinTek Explorer.
14. To simplify the mechanism, we assume here that chemical
cleavage and P i release are both irreversible. Regarding P i
release, this is the case for most enzymes, since P i affinity is
generally very low. In addition, free P i is trapped by the binding
to MDCC-PBP making the reaction quasi-irreversible. In
314
Simone Kunzelmann