KINETIC OPTIMIZATION OF BACTERIORHODOPSIN BY ASPARTIC ACID-96 AS AN INTERNAL PROTON DONOR

The reprotonation of the Schiff base in bacteriorhodopsin (M-decay) was measured in water and deuterium oxide as a function of pH (pD) and ionic strength. Comparison of the wild-type and two mutated proteins (D96 → N or G) clearly demonstrated the function of aspartic acid 96 as an internal protein proton donor for this reaction. Its removal lowers the Arrhenius activation energy of the reaction from 75 kJ/mol to 32 kJ/mol (D96 → N) and 18 kJ/mol (D96 → G) at pH 7 and 10 mM ionic strength. Concomitant with this change is a drastic decrease in rate constant from 166 s-1 to 0.4 s-1 (D96 → N) and 5 s-1 (D96 → G) at pH 7 and 20°C. The effect is due to a small decrease in the free energy of activation but due mainly to a large decrease in the entropy of activation of the mutant proteins. This exemplifies clearly the advantage of a spatially fixed proton donor selected by the protein structure. Lack of aspartic acid 96 also renders the proton pump dependent on external pH (physiologically important) and ionic strength (of no physiological importance). The influences of both parameters on the reaction kinetics can be explained if pH-induced surface potential and surface charge changes at the membrane-bulk interface are taken into account. © 1990.