Mechanism of the reaction catalyzed by acetoacetate decarboxylase. Importance of lysine 116 in determining the pK(a) of active-site lysine 115

Acetoacetate decarboxylase from Clostridium acetobutylicum (AAD) catalyzes the decarboxylation of acetoacetate via a Schiff base intermediate [Hamilton, G. A., & Westheimer, F. H. (1959) J. Am. Chem. Sec. 81, 6332; Fridovich, I., & Westheimer F. H. (1962) J. Am. Chem. Sec. 84, 3208]. The pK(a) of the active-site lysine (Lys 115) is 6.0, 4.5 pK(a) units less than the pK(a) of lysine in solution [Kokesh, F. C., & Westheimer, F. H. (1971) J. Am. Chem. Sec. 93, 7270; Frey, P. A., Kokesh, F. C., & Westheimer, F. H. (1971) J. Am. Chem. Sec. 93, 7266; Schmidt, D. E., Jr., & Westheimer, F. H. (1971) Biochemistry 10, 1249]. Westheimer and co-workers hypothesized that the pK(a) of Lys 115 is decreased by its spatial proximity to the E-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-bromoethylammonium 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 pK(a) of Lys 115 in the mutant AADs were evaluated following imine formation with 5-nitrosalicylaldehyde and reduction with NaBH4. Whereas the pK(a) of Lys 115 in K116R is similar to that observed for wild type AAD, the pK(a)s 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 pK(a) of Lys 115 to 5.9. These data support Westheimer's hypothesis that the pK(a) of the Schiff base-forming Lys 115 is decreased by its spatial proximity to the epsilon-ammonium group of Lys 116.