DAP Epimerase

In addition to n-alanine and n-glutamate, many bacterial cell walls also contain meso-diaminopimelate (DAP) [2]. DAP is produced by epimerization from l,l-DAP to d,l-DAP by the cofactor independent diaminopimelate epimerase [97, 98]. The structure of this enzyme has been solved and two cysteines in the active site were proposed to be the acid-base catalysts [99]. The pattern of label incorporation from tritiated water is consistent with a two-base mechanism [97]. The enzyme has been shown to be stoichiometrically inhibited by the thiol alkylating agent aziDAP [97]. Interestingly, DAP epimerase has an equilibrium constant of 2 (Keq = [d,l]/[l,l]) duc to the statistically expected higher concentration of the [d,l] form at equilibrium between these species [100]. 

DAP-epimerase yields an unusual overshoot pattern a normal overshoot is seen in the l,l —> d,l direction, but an unprecedented double-overshoot is seen in the D,L —> L,L direction [100]. A simulation (using the program DynaFit [101]) of the DAP-epimerase double overshoot, based on rate constant values used in simulations by Koo and Blanchard, is shown in Fig. 7.17A. Koo and Blanchard proposed that the double overshoot is due to the fact that two stereocenters undergo exchange, but only one is racemized. The full reaction scheme, as presented by Koo and Blanchard, is illustrated in Fig. 7.18. The D,L-substrate initially reacts faster than the L,L-substrate, and enters an isotopically sensitive branch point. One observes a classic overshoot in both directions due to the fact that the substrate-derived KIE for the reverse direction results in a transient accumulation of the product (the orthodox source of an overshoot). However, the additional overshoot in the d,l —> l,l direction was attributed to accumulation of [ H]-d,[ H]-l-DAP in the isotopically sensitive branch pathway, which results in a transient accumulation of the D,L-isomer, even though the reaction commenced with [ H]-d,[ H]-l-isomer (i.e., in the opposite direction from an orthodox overshoot). Surprisingly, removal of the isotopically sensitive branch point, such that only the bold species in Fig. 7.18 are present, yields an effectively identical simulated double overshoot... 

Figure 7.17. A, Simulated double overshoot for DAP-epimerase using the program DynaFit [101] and the rate constant values from Koo and Blanchard [100]. B, Simplified simulated double overshoot, in A/hich the isotopically sensitive branch path A/ay from Fig. 7.18 is removed.

Figure 7.18. Kinetic scheme for DAP-epimerase, as described in Koo and Blanchard [100], used to generate the simulated overshoot in Fig. 7.17A. The species in bold type represent the simplified overshoot used to generate simulated overshoot in Fig. 7.17B.

Figure 7.19. Concentrations of products, substrates and intermediates from the double overshoot of DAP-epimerase. The time points that correspond to the peaks of the two overshoots of the double overshoot are indicated.

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