Hydrazine and amphetamine binding to amine oxidases: old drugs with new prospects.

Tranylcypromine (TCP), an amphetamine, is a reversible inhibitor of copper-containing amine oxidases. We have solved the structure of the complex of TCP with the amine oxidase from E. coli (ECAO) and shown that only the (+)-enantiomer of TCP binds. Kinetic studies on 2-phenylethylamine and TCP binding to wild-type ECAO and mutational variants fully support the model in which binding of the protonated amine is the first step in the catalytic cycle. Hydrazines are irreversible inhibitors of copper-containing amine oxidases. Binding of hydrazines leads to an adduct ("Adduct 1") with a chromophore at 430 nm which converts at higher pH to another adduct ("Adduct 2") with a chromophore at 520 nm. We have determined the structures of Adduct 1 and 2 for 2-hydrazinopyridine reacted with ECAO. It has been found that Adduct 1 corresponds to the hydrazone and azo tautomers whilst Adduct 2 corresponds to the azo tautomer coordinated to the active site copper. The implications of these results in developing more specific drugs are discussed.

Conserved tyrosine-369 in the active site of Escherichia coli copper amine oxidase is not essential.

Copper amine oxidases are homodimeric enzymes that catalyze two reactions: first, a self-processing reaction to generate the 2,4,5-trihydroxyphenylalanine (TPQ) cofactor from an active site tyrosine by a single turnover mechanism; second, the oxidative deamination of primary amine substrates with the production of aldehyde, hydrogen peroxide, and ammonia catalyzed by the mature enzyme. The importance of active site residues in both of these processes has been investigated by structural studies and site-directed mutagenesis in enzymes from various organisms. One conserved residue is a tyrosine, Tyr369 in the Escherichia coli enzyme, whose hydroxyl is hydrogen bonded to the O4 of TPQ. To explore the importance of this site, we have studied a mutant enzyme in which Tyr369 has been mutated to a phenylalanine. We have determined the X-ray crystal structure of this variant enzyme to 2.1 A resolution, which reveals that TPQ adopts a predominant nonproductive conformation in the resting enzyme. Reaction of the enzyme with the irreversible inhibitor 2-hydrazinopyridine (2-HP) reveals differences in the reactivity of Y369F compared with wild type with more efficient formation of an adduct (lambda(max) = 525 nm) perhaps reflecting increased mobility of the TPQ adduct within the active site of Y369F. Titration with 2-HP also reveals that both wild type and Y369F contain one TPQ per monomer, indicating that Tyr369 is not essential for TPQ formation, although we have not measured the rate of TPQ biogenesis. The UV-vis spectrum of the Y369F protein shows a broader peak and red-shifted lambda(max) at 496 nm compared with wild type (480 nm), consistent with an altered electronic structure of TPQ. Steady-state kinetic measurements reveal that Y369F has decreased catalytic activity particularly below pH 6.5 while the K(M) for substrate beta-phenethylamine increases significantly, apparently due to an elevated pK(a) (5.75-6.5) for the catalytic base, Asp383, that should be deprotonated for efficient binding of protonated substrate. At pH 7.0, the K(M) for wild type and Y369F are similar at 1.2 and 1.5 microM, respectively, while k(cat) is decreased from 15 s(-1) in wild type to 0.38 s(-1), resulting in a 50-fold decrease in k(cat)/K(M) for Y369F. Transient kinetics experiments indicate that while the initial stages of enzyme reduction are slower in the variant, these do not represent the rate-limiting step. Previous structural and solution studies have implicated Tyr369 as a component of a proton shuttle from TPQ to dioxygen. The moderate changes in kinetic parameters observed for the Y369F variant indicate that if this is the case, then the absence of the Tyr369 hydroxyl can be compensated for efficiently within the active site.

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 coliamine oxidase (ECAO) is the catalytic base which performs the key step of proton abstraction. This paper explores in more depth this and other roles of Asp383. The crystal structures of three mutational variants are presented together with their catalytic properties, visible spectra, and binding properties for a substrate-like inhibitor, 2-hydrazinopyridine (2-HP), in comparison 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 substrate binding position (C5) than for TPQ ring carbons C1-C4. A role of Asp383, whose carboxylate is located close to O5, is to stabilize the TPQ in its major conformation in the pocket. Replacement of Asp383 with the isostructural, but chemically distinct, Asn383 does not affect the location or dynamics of the TPQ cofactor significantly, but eliminates catalytic activity and drastically reduces the affinity for 2-HP. Removal of the side chain carboxyl moiety, as in Ala383, additionally allows the TPQ the greater conformational flexibility to coordinate to the copper, which demonstrates that Asp383 helps maintain the active site structure by preventing TPQ from migrating to the copper. Glu383 has a greatly decreased catalytic activity, as well 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 pivotal motion of the TPQ by hindering its movement within the wedge-shaped active site pocket. The results show that Asp383 performs multiple roles in the 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 mobility of the TPQ that is essential to catalysis.