Nucleotide Binding Assay to MalFGK2
where Fmax is the maximal subtracted fluorescence at saturating amount of TNP-ATP and Kd is the equilibrium dissociation constant of TNP-ATP for MalFGK2. To determine the affinity of MalFGK2 for ATP, TNP-ATP (80 μm) was incubated with MalFGK2 nanodiscs (2 μm) for 5 min at 25 °C and then titrated with an increased amount of ATP. The fluorescence data were fit to Equation 2,
in which F0 is the fluorescence value in the absence of ATP, F1 is the fluorescence value in the presence of saturating amount of ATP, [I] is the ATP concentration, and Ki,app is the apparent inhibition constant of ATP at the specified amount of TNP-ATP. The Kd,app of ATP for MalFGK2 is calculated from Ki,app by Equation 3,
in which [L] is the TNP-ATP concentration, and Kd is the dissociation constant of TNP-ATP for MalFGK2.
where A is the amount of calculated ADP, A0 is the amount of ADP in the exponential phase, kburst is the rate constant for the burst phase, and klinear is the rate constant for the slower linear phase.
where A0 is the amount of ATP before the initiation of the reaction, B is the amount of ATP at the end of the burst phase of the reaction, kburst is the rate constant for the burst phase, and klinear is the rate constant for the slower linear phase.
Binding of ATP to MalK Is Independent of MalE and Maltose
|Km for ATP||Kd for TNP-ATP||Kd,app for ATP|
|MalFGK2||233 ± 42||9 ± 1||211 ± 23|
|MalFGK2, MalE||274 ± 32||10 ± 2||194 ± 22|
|MalFGK2, MalE, maltose||283 ± 41||9 ± 1||224 ± 33|
MalE Stimulates the ATP Cleavage Reaction
Release of Pi Limits the Kinetics of the ATPase Cycle
|MalFGK2||0.3 ± 0.1||NA||NA|
|MalFGK2, MalE||1.2 ± 0.2||60 ± 10||69 ± 2|
|MalFGK2, MalE, maltose||7.8 ± 0.3||70 ± 15||66 ± 1|
|MalF500||27.5 ± 0.4||90 ± 20||68 ± 1|
|MalFGK2, MalE, maltose, EIIAGlc||1.8 ± 0.2||4 ± 2||5.4 ± 0.3|
MalF500 Hydrolyzes ATP Constitutively
The Regulator EIIAGlc Inhibits the Cleavage of ATP
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