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Biochemistry. 1999 Apr 6;38(14):4416-22.

Chemical mechanism of Haemophilus influenzae diaminopimelate epimerase.

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  • 1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.


Seven unique enzymatic steps lead to the biosynthesis of L-lysine from L-aspartate semialdehyde and pyruvate in bacteria. The immediate precursor to L-lysine is D,L-diaminopimelate, a diamino acid which is incorporated into the pentapeptide of the Gram-negative peptidoglycan moiety. D,L-Diaminopimelate is generated from the corresponding L,L-isomer by the dapF-encoded epimerase. Diaminopimelate epimerase is a representative of the pyridoxal phosphate-independent amino acid racemases, for which substantial evidence exists supporting the role of two cysteine residues as the catalytic acid and base. The pH dependencies of the maximum velocities in the L,L --> D,L and D,L --> L,L direction depend on a single catalytic group exhibiting pK values of 7.0 and 6.1, respectively, which must be unprotonated for activity. The pH dependencies of the V/K values in both directions depend on the ionization of two groups, one exhibiting a pK value of 6.7 which must be unprotonated and one exhibiting a pK value of 8.5 which must be protonated. Primary kinetic isotope effects on V and V/K are unequal, with D(V/K) being larger than DV in both the forward and reverse directions. Solvent kinetic isotope effects in both directions are inverse on V/K, but normal on V. Both of these isotopic observations support a model in which proton isomerization after catalysis and substrate dissociation is kinetically significant. A single solvent "overshoot" is observed when L, L-diaminopimelate is incubated with enzyme in D2O; however, an unprecedented double overshoot is observed when D,L-diaminopimelate is incubated with enzyme in D2O. A model has been developed which allows these two overshoots to be simulated. A chemical mechanism is proposed invoking the function of two cysteine residues, Cys73 and Cys217, observed in the recently determined three-dimensional structure of this enzyme [Cirilli, M., et al. (1998) Biochemistry 37, 16452-16458], as the acid and base in the mechanism.

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