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 recombinant 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.