The incorporation of D-alanine from L-alanine into the peptidoglycan precursor, uridine diphosphate-N-acetylmuramyl-L-Ala-D-γ-Glu-L-Lys-D-Ala-D-Ala, is catalyzed by the sequential action of alanine racemase (1); D-alanine: D-alanine ligase (adenosine diphosphate) (2); and uridine diphosphate-N-acetylmuramyl-L-Ala-D-γ-Glu-L-Lys: D-Ala-D-Ala ligase (adenosine diphosphate) (3). The product of reaction 2, D-Ala-D-Ala, is a competitive inhibitor (Ki = 1.2 × 10-3 M) of D-alanine:D-alanine ligase. The specificity of product inhibition differs from that predicted from substrate specificity studies; e.g., D-Ala-D-norval, D-Ala-D-α-amino-n-butyric acid (Ki) = 6.0 × 10-4 M) > D-Ala-D-Ala > D-Ala-D-Ser > D-Ala-D-Thr > D-α-amino-n-butyryl-D-α-amino-n-butyric acid (Ki) = 5.3 × 10-3 M), D-Ala-D-Val D-α-amino-n-butyryl-D-Ala. A kinetic analysis of the inhibition is consistent with a model that assumes at least two binding sites for product: i.e., EA, EAA, EP, EAP, and EAP2, where A = D-alanine and P = D-Ala-D-Ala. An analysis of the inhibition by D-Ala-D-norval with the Hill equation shows a change in the interaction coefficient from n = 1 to n = 3 as the concentration of D-Ala-D-norval is increased. These results suggest the presence of more than one binding site for D-Ala-D-Ala in D-alanine:D-alanine ligase. Since Keq for alanine racemase is 1, the inhibition of D-alanine: D-alanine ligase (adenosine diphosphate) by D-Ala-D-Ala would, in effect, control the utilization of L-alanine by the racemase. Further, the level of D-Ala-D-Ala available to enzyme (3) may control the rate of uridine diphosphate-N-acetylmuramylpentapeptide formation. © 1969, American Chemical Society. All rights reserved.
