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) catal yzes the decarboxylation of acetoacetate via a Schiff base intermediat e [Hamilton, G. A., & Westheimer, F. H. (1959) J. Am. Chem. Sec. 81, 6 332; Fridovich, I., & Westheimer F. H. (1962) J. Am. Chem. Sec. 84, 32 08]. The pK(a) of the active-site lysine (Lys 115) is 6.0, 4.5 pK(a) u nits less than the pK(a) of lysine in solution [Kokesh, F. C., & Westh eimer, 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]. Westhei mer 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 11 5 and Lys 116. Two substitutions for Lys 115 (K115C and K115Q) were bo th catalytically inactive at pH 5.95, the pH optimum of wild type AAD, demonstrating the importance of this residue in catalysis. Activity c ould 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 followi ng imine formation with 5-nitrosalicylaldehyde and reduction with NaBH 4. Whereas the pK(a) of Lys 115 in K116R is similar to that observed f or wild type AAD, the pK(a)s of Lys 115 in K116C and K116N were elevat ed to > 9.2. Alkylation of Cys 116 in K116C with 2-BEAB resulted in bo th 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 S chiff base-forming Lys 115 is decreased by its spatial proximity to th e epsilon-ammonium group of Lys 116.