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Acetoacetate decarboxylase from Clostridium acetobutylicum (AAD) catalyzes the decarboxylation of acetoacetate via a Schiff base intermediate Hamilton, G. A., Fridovich, I., Frey, P. A., Kokesh, F. C., Schmidt, D. E., Jr., & Westheimer, F. H. (1971) Biochemistry 10, 1249. Westheimer and co-workers hypothesized that the pKa of Lys 115 is decreased by its spatial proximity to the epsilon-ammonium group of Lys 116. We have investigated this proposal by studying site-directed mutants of Lys 115 and Lys 116. Two substitutions for Lys 115 (K115C and K115Q) were both catalytically inactive at pH 5.95, the pH optimum of wild type AAD, demonstrating the importance of this residue in catalysis. Activity could be restored to K115C by aminoethylation with 2-bromoethyl-ammonium bromide (2-BEAB). Substitutions for Lys 116 (K116C, K116N, and K116R) had reduced but significant activities at pH 5.95. The effects of Lys 116 on the pKa of Lys 115 in the mutant AADs were evaluated following imine formation with 5-nitrosalicylaldehyde and reduction with NaBH4. Whereas the pKa of Lys 115 in K116R is similar to that observed for wild type AAD, the pKaS of Lys 115 in K116C and K116N were elevated to > 9.2. Alkylation of Cys 116 in K116C with 2-BEAB resulted in both significant activation and restoration of the pKa of Lys 115 to 5.9. These data support Westheimer's hypothesis that the pKa of the Schiff base-forming Lys 115 is decreased by its spatial proximity to the epsilon-ammonium group of Lys 116.
Highbarger et al. (Mon,) studied this question.