Copp
er amin
e oxidas
es ar
e homodim
eric
enzym
es that catalyz
e two r
eactions: first, a s
elf-proc
essing r
eaction to g
en
erat
e th
e 2,4,5-trihydroxyph
enylalanin
e (TPQ) cofactor from an acti
ve sit
e tyrosin
eby a singl
e turno
ver m
echanism; s
econd, th
e oxidati
ve d
eamination of primary amin
e substrat
es with th
eproduction of ald
ehyd
e, hydrog
en p
eroxid
e,
and ammonia catalyz
ed by th
e matur
e enzym
e. Th
e importanc
eof acti
ve sit
e r
esidu
es in both of th
es
e proc
ess
es has b
een in
vestigat
ed by structural studi
es
and sit
e-dir
ect
ed mutag
en
esis in
enzym
es from
various organisms. On
e cons
er
ved r
esidu
e is a tyrosin
e, Tyr369 inth
e Escherichia coli enzym
e, whos
e hydroxyl is hydrog
en bond
ed to th
e O4 of TPQ. To
explor
e th
eimportanc
e of this sit
e, w
e ha
ve studi
ed a mutant
enzym
e in which Tyr369 has b
een mutat
ed to aph
enylalanin
e. W
e ha
ve d
et
ermin
ed th
e X-ray crystal structur
e of this
variant
enzym
e to 2.1 Å r
esolution,which r
eveals that TPQ adopts a pr
edominant nonproducti
ve conformation in th
e r
esting
enzym
e. R
eactionof th
e enzym
e with th
e irr
eversibl
e inhibitor 2-hydrazinopyridin
e (2-HP) r
eveals diff
er
enc
es in th
e r
eacti
vityof Y369F compar
ed with wild typ
e with mor
e effici
ent formation of an adduct (
![](/imag<font color=)
es/gifchars/lambda.gif" BORDER=0 >
max = 525 nm) p
erhapsr
efl
ecting incr
eas
ed mobility of th
e TPQ adduct within th
e acti
ve sit
e of Y369F. Titration with 2-HP alsor
eveals that both wild typ
e and Y369F contain on
e TPQ p
er monom
er, indicating that Tyr369 is not
ess
ential for TPQ formation, although w
e ha
ve not m
easur
ed th
e rat
e of TPQ biog
en
esis. Th
e UV-
vissp
ectrum of th
e Y369F prot
ein shows a broad
er p
eak
and r
ed-shift
ed
![](/imag<font color=)
es/gifchars/lambda.gif" BORDER=0 >
max at 496 nm compar
ed with wildtyp
e (480 nm), consist
ent with an alt
er
ed
el
ectronic structur
e of TPQ. St
eady-stat
e kin
etic m
easur
em
entsr
eveal that Y369F has d
ecr
eas
ed catalytic acti
vity particularly b
elow pH 6.5 whil
e th
e KM for substrat
e![](/imag<font color=)
es/gifchars/b
eta2.gif" BORDER=0 ALIGN="middl
e">-ph
en
ethylamin
e incr
eas
es significantly, appar
ently du
e to an
el
evat
ed p
Ka (5.75-6.5) for th
e catalyticbas
e, Asp383, that should b
e d
eprotonat
ed for
effici
ent binding of protonat
ed substrat
e. At pH 7.0, th
eKM for wild typ
e and Y369F ar
e similar at 1.2
and 1.5
![](/imag<font color=)
es/
entiti
es/mgr.gif">M, r
esp
ecti
vely, whil
e kcat is d
ecr
eas
ed from 15s
-1 in wild typ
e to 0.38 s
-1, r
esulting in a 50-fold d
ecr
eas
e in
kcat/KM for Y369F. Transi
ent kin
etics
exp
erim
ents indicat
e that whil
e th
e initial stag
es of
enzym
e r
eduction ar
e slow
er in th
e variant, th
es
e donot r
epr
es
ent th
e rat
e-limiting st
ep. Pr
evious structural
and solution studi
es ha
ve implicat
ed Tyr369 as acompon
ent of a proton shuttl
e from TPQ to dioxyg
en. Th
e mod
erat
e chang
es in kin
etic param
et
ers obs
er
vedfor th
e Y369F
variant indicat
e that if this is th
e cas
e, th
en th
e abs
enc
e of th
e Tyr369 hydroxyl can b
ecomp
ensat
ed for
effici
ently within th
e acti
ve sit
e.