His‐163 is a stereospecific proton donor in the mechanism of d‐glucosaminate‐6‐phosphate ammonia‐lyase

d‐Glucosaminate‐6‐phosphate ammonia‐lyase (DGL) catalyzes the conversion of d‐glucosaminate‐6‐phosphate to 2‐keto‐3‐deoxyglutarate‐6‐phosphate, with stereospecific protonation of C‐3 of the product. The crystal structure of DGL showed that His‐163 could serve as the proton donor. H163A mutant DGL is fully active in the steady‐state reaction, and the pre‐steady‐state kinetics are very similar to those of wild‐type DGL. However, H163A DGL accumulates a transient intermediate with λ(max) at 293 nm during the reaction that is not seen with wild‐type DGL. Furthermore, NMR analysis of the reaction of H163A DGL in D(2)O shows that the product is a mixture of deuterated diastereomers at C‐3. These results establish that His‐163 is the proton donor in the reaction mechanism of DGL.