Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the Haemophilus influenzae dapE-encoded desuccinylase: Metal activation, solvent isotope effects, and kinetic mechanism

Timothy L. Born, Renjian Zheng, John S. Blanchard

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

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 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 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 (D2O)V/K of 0.62 and (D2O)V 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.

Original languageEnglish (US)
Pages (from-to)10478-10487
Number of pages10
JournalBiochemistry
Volume37
Issue number29
DOIs
StatePublished - Jul 21 1998

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Diaminopimelic Acid
Haemophilus influenzae
Isotopes
Hydrolysis
Metals
Chemical activation
Kinetics
Enzymes
Sequence Homology
Stoichiometry
Escherichia coli
Lysine
Metal ions
Zinc
Binding Sites
Ions
Substrates
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the Haemophilus influenzae dapE-encoded desuccinylase : Metal activation, solvent isotope effects, and kinetic mechanism. / Born, Timothy L.; Zheng, Renjian; Blanchard, John S.

In: Biochemistry, Vol. 37, No. 29, 21.07.1998, p. 10478-10487.

Research output: Contribution to journalArticle

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