HYDROLYSIS OF N-SUCCINYL-L,L-DIAMINOPIMELIC ACID BY THE HAEMOPHILUS-INFLUENZAE DAPE-ENCODED DESUCCINYLASE - METAL ACTIVATION, SOLVENT ISOTOPE EFFECTS, AND KINETIC MECHANISM

Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the dapE-encoded d esuccinylase is required for the bacterial synthesis of lysine and mes o-diaminopimelic acid, We have investigated the catalytic mechanism of the recombinant enzyme from Haemophilus influenzae. The desuccinylase was overexpressed in Escherichia coli and purified to homogeneity. St eady-state kinetic experiments verified that the enzyme is metal-depen dent, with a K-m, for N-succinyl-L,L-diaminopimelic acid of 1.3 mM and a turnover number of 200 s(-1) in the presence of zinc. The maximal v elocity was independent of pH above 7 bur decreased with a slope of 1 below pH 7. The pH dependence of V/K was bell-shaped with apparent pKs of 6.5 and 8.3. Both L,L- and D,L-diaminopimelic acid were competitiv e inhibitors of the substrate, but D,D-diaminopimelic acid was not. So lvent kinetic isotope effect studies yielded inverse isotope effects, with values for V-D2O/K of 0.62 and V-D2O of 0.78. Determination of me tal stoichiometry by ICP-AES indicated one tightly bound metal ion, wh ile sequence homologies suggest the presence of two metal binding site s. On the basis of these observations, we propose a chemical mechanism for this metalloenzyme, which has a number of important structurally defined homologues.