Arg302 governs the pK a of Glu325 in LacY

Significance The alkaline pK for galactoside binding by the lactose permease of Escherichia coli correlates precisely with the pK a of Glu325, as determined by reaction-induced surface-enhanced infrared absorption spectroscopy (SEIRAS). Glu325 must be protonated for LacY to bind sugar effectively, but deprotonation is also essential for transport. SEIRAS is utilized to test the effect of mutating residues in the immediate neighborhood of Glu325 based on the rationale that interaction will alter the pK a . Neutral replacement of Arg302 with Ala has little or no effect, while replacement with positively charged Lys causes a two-pH unit acid shift. Since a number of other mutations in the vicinity have little effect, it is concluded that Arg302 is important for deprotonation of Glu325.