Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study

L‐Arginine:glycine amidinotransferase catalyzes the committed step in the biosynthesis of creatine. Eight active‐site mutants, D170N, D254N, H303V, D305A, R322E, S355A, C407S, and C410A of recom‐binant human L‐arginine : glycine amidinotransferase were prepared by site‐directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants D170N, D254N, and C407S have been determined at 0.28‐nm, 0.29‐nm and 0.236‐nm resolution, respectively. The mutation of active‐site residues which are involved in substrate‐binding yielded inactive mutants. Substitution of Asp254, which is not directly involved in substrate binding but is thought to transfer protons in concert with the His303 imidazole group, results in a strongly (2000‐fold) reduced activity. However, the substitution of Cys410, a residue near the active site but not involved in catalysis or substrate binding, by Ala does not change the kinetic properties with respect to the wild‐type enzyme. The loss of enzymatic activity of the D170N, D254N, C407S and likely all other mutants is solely due to the inserted point mutations, affecting substrate binding or transition‐state stabilization, and not due to major conformational rearrangements of the protein. These results show that a His‐Asp pair on one side of the substrate and a Cys on the other side are key residues for activity and are part of a disjoint triad. The imidazole ring of the His is proposed to act as a general acid/general base during catalysis whereas the Cys acts as a nucleophile analogous to Cys25 of papain‐like cysteine proteinases.