The active site base controls cofactor reactivity in Escherichia coli amine oxidase: X-ray crystallographic studies with mutational variants

Amine oxidases utilize a proton abstraction mechanism following binding of the amine substrate to the C5 position of the cofactor, the quinone form of trihydroxyphenylalanine (TPQ). Previous work [Wilmot, C. M,, et al. (1997) Biochemistry 36, 1608-1620] has shown that Asp383 in Escherichia coli amin e oxidase (ECAO) is the catalytic base which performs the key step of proto n abstraction. This paper explores in more depth this and other roles of As p383, The crystal structures of three mutational variants are presented tog ether with their catalytic properties, visible spectra, and binding propert ies for a substrate-like inhibitor, 2-hydrazinopyridine (2-HP), in comparis on to those of the wild type enzyme. In wild type ECAO, the TPQ is located in a wedge-shaped pocket which allows more freedom of movement at the subst rate binding position (C5) than for TPQ ring carbons C1-C4, A role of Asp38 3, whose carboxylate is located close to O5, is to stabilize the TPQ in its major conformation in the pocket. Replacement of Asp383 with the isostruct ural, but chemically distinct, Asn383 does not affect the location or dynam ics of the TPQ cofactor significantly, but eliminates catalytic activity an d drastically reduces the affinity for 2-HP. Removal of the side chain carb oxyl moiety, as in Ala383, additionally allows the TPQ the greater conforma tional flexibility to coordinate to the copper, which demonstrates that Asp 383 helps maintain the active site structure by preventing TPQ from migrati ng to the copper. Glu383 has a greatly decreased catalytic activity, as wel l as a decreased affinity for 2-HP relative to that of wild type ECAO. The electron density reveals that the longer side chain of Glu prevents the piv otal motion of the TPQ by hindering its movement within the wedge-shaped ac tive site pocket. The results show that Asp383 performs multiple roles in t he catalytic mechanism of ECAO, not only in acting as the active site base at different stages of the catalytic cycle but also in regulating the mobil ity of the TPQ that is essential to catalysis.