1. SLIDE SHOW II
E325 Mutants

2. H+ IX R302 + - E325 X H322 VIII K319 - E269 + - D240 R + C 144 148 VII
A. Ground state IX
R302 + -
E325 X
VIII
H322 H+
K319 -
E269 + -
D240 R + C
144
148
VII - V
E126 IV

3. H+ IX R302 + A325 X H322 VIII K319 - E269 + - D240 R + C 144 148 VII -
B. Mutant E325A does exchange and counterflow, but no reactions involving H+ translocation IX
R302 +
A325 X
VIII
H322 H+
K319 -
E269 + -
D240 R + C
144
148
VII - V
E126 IV

4. C. Mutant E325D is partially uncoupled.IX
R302 + -
D325 X
VIII
H322
H+o
K319 -
E269 + -
D240 R + C
144
148
VII - V
E126 IV

5. D. Substrate binds from the outside with lower affinity.IX
R302 + -
D325 X
VIII
H322
H+o
K319 -
E269 + -
D240 R + C
144
148 So
VII - V
E126 IV

6. E. Why E325D has lower affinity for ligand

7. H+o IX R302 + - D325 X H322 VIII K319 - E269 + - D240 R + C 144 148
F. Binding of substrate causes a conformational change disrupting the interaction of Glu269 and His322, ….
K319
H322
VIII +
R302 IX X
D325 - C R
148
144 V
E269
H+o - + -
D240
E126 IV
VII + So -

8. H+ IX R302 + - VIII D325 H322 X K319 E269 + - - D240 R + C 144 148 VII
G. ... causes substrate to become accessible to the internal surface, and the proton moves to His322/Asp325. IX
R302 + - H+
VIII
D325
H322 X
K319
E269 - + -
D240 R + C
144
148 Si
VII - V
E126 IV

9. H+ IX R302 + - VIII D325 H322 X K319 E269 + - - D240 R + C 144 148 VII
H. Rotation of Helix X continues towards the hydrophobic phase of the membrane, IX
R302 + - H+
VIII
D325
H322 X
K319
E269 - + -
D240 R + C
144
148 Si
VII - V
E126 IV

10. I. Substrate dissociates to the inside.IX H+
R302 - +
VIII
D325
H322
K319
E269 X + - - Si
D240 R + C
144 Si
148
VII - V
E126 IV

11. J. Asp325 re-juxtaposes with Arg302,IX H+
R302 - +
VIII
D325
H322
K319
E269 X + - -
D240 R + C
144
148
VII - V
E126 IV

12. K. and the proton is released to the inside with decreased efficiency.IX
R302 + -
D325 X
H+i
VIII
H322
K319 -
E269 + -
D240 R + C
144
148
VII - V
E126 IV

13. H+o IX R302 + - D325 X H322 VIII K319 - E269 + - D240 R + C 144 148
L. The permease relaxes into the ground state and becomes protonated from the outside. IX
R302 + -
D325 X
VIII
H322
H+o
K319 -
E269 + -
D240 R + C
144
148
VII - V
E126 IV

14. M. Like Glu325 mutants, R302S [or R302A]
permease is specifically defective in
lactose/H+ symport, but catalyzes exchange 10
100
Lactose in (%) 20 40 60 80
120
Time (sec)
Efflux
Exchange