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 desuccinylase is required for the bacterial synthesis of lysine and meso-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. Steady-state kinetic experiments verified that the enzyme is metal-dependent, with a Km 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 velocity was independent of pH above 7 but 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 competitive inhibitors of the substrate, but d,d-diaminopimelic acid was not. Solvent kinetic isotope effect studies yielded inverse isotope effects, with values for D2OV/K of 0.62 and D2OV of 0.78. Determination of metal stoichiometry by ICP-AES indicated one tightly bound metal ion, while sequence homologies suggest the presence of two metal binding sites. On the basis of these observations, we propose a chemical mechanism for this metalloenzyme, which has a number of important structurally defined homologues.
Born
T L
TL
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Zheng
R
R
Blanchard
J S
JS
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D003035:Cobalt / Q000378:metabolism*; D017666:Deuterium Oxide / Q000378:metabolism*; D003960:Diaminopimelic Acid / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012997:Solvents; D019802:Succinic Acid / Q000378:metabolism / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism*
D000581:Amidohydrolases; D003035:Cobalt; D017666:Deuterium Oxide; D003960:Diaminopimelic Acid; D011994:Recombinant Proteins; D012997:Solvents; D019802:Succinic Acid; D015032:Zinc; C054685:N-succinyl-l,l-diaminopimelic acid desuccinylase