Conserved tyrosine-369 in the active site of Escherichia coli copper amineoxidase is not essential

Copper amine oxidases are homodimeric enzymes that catalyze two reactions: first, a self-processing reaction to generate the 2,4,5-trihydroxyphenylala nine (TPQ) cofactor from an active site tyrosine by a single turnover mecha nism; second, the oxidative deamination of primary amine substrates with th e production of aldehyde, hydrogen peroxide, and ammonia. catalyzed by the mature enzyme. The importance of active site residues in both of these proc esses has been investigated by structural studies and site-directed mutagen esis 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 hav e determined the X-ray crystal structure of this variant enzyme to 2.1 Angs trom resolution, which reveals that TPQ adopts a predominant nonproductive conformation in the resting enzyme. Reaction of the enzyme with the irrever sible inhibitor 2-hydrazinopyridine (2-HP) reveals differences in the react ivity of Y369F compared with wild type with more efficient formation of an adduct (lambda (max) = 525 nm) perhaps reflecting. increased mobility of th e TPQ adduct within the active site of Y369F. Titration with 2-HP also reve als that both wild type and Y369F contain one TPQ per monomer, indicating t hat Tyr369 is not essential for TPQ formation, although we have not measure d the rate of TPQ biogenesis. The UV-vis spectrum of the Y369F protein show s 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 Stead y-state kinetic measurements reveal that Y369F has decreased catalytic acti vity particularly below pH 6.5 while the K-M for substrate beta -phenethyla mine 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 Y369 F are similar at 1.2 and 1.5 muM, respectively, while k(cat) is decreased f rom 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 t he initial stages of enzyme reduction are slower in the variant, these do n ot represent the rate-limiting step. Previous structural and solution studi es have implicated Tyr369 as a component of a proton shuttle from TPQ to di oxygen. The moderate changes in kinetic parameters observed for the Y369F v ariant indicate that if this is the case, then the absence of the Tyr369 hy droxyl can be compensated for efficiently within the active site.