Spectroscopic studies of the active sites in copper-containing amine oxidases

Pharmaco~g~alReseamh Communication~VoL 2~ Supp~mentl~ 1988

151

SPECTROSCOPIC STUDIES OF THE ACTIVE SITES IN COPPER-CONTAINING AMINE OXIDASES

D, M. Dooley Department of Chemistry, Amherst College, Amherst, MA 01002, USA Key Words: Copper/Pyrroloquinoline q u i n o n e A variety of spectroscopic methods have been used to probe the copper sites, interactions plasma,

the organic cofactor, and the copper-cofactor in amine oxidases isolated from bovine plasma, porcine

porcine kidney, pea seedlings,

and Arthrobacter P[.

Th~

magnetic field dependence of the solvent HtO T, (the nuclear magnetic relaxation dispersion, NMRD) has been measured for several amine oxidases;

in all cases the NMRD results are consistent with the

presence of at least one coordinated Ht0 that is in rapid exchange with solvent.

Since the paramagnetic relaxivity is relatively

insensitive to anion binding, which is known to occur equatorially (McCracken et al. ([g87)), an ax£aiiy coordinated H20 ~s probably also present.

The catalytic activity,

the 480 nm absorbance, and the NMRD

profiles of porcine plasma and bovine plasma amine oxidase are directly proportional

to the copper content.

Hence the two copper

sites seem to have similar accessibility to solvent and to contribute

nrlllnlTV rn r n r ~ I v ~ i R .

Thp r n ~ e ~ n n ~

oF P-pyridv~hydrazi~,~ w~lh r l a l { v n

and metal depleted forms of various amine oxidases have been monitored by absorption and resonance Raman spectroscopoy.

Initially a 460 ~11~I

(410 nm in the apo proteins) chromophore is produced, which may be converted to a 520 nm chromophore only when copper is present.

The

extinction coefficients are consistent w~th the presence of two reactive carbonyl groups per enzyme molecule.

Figure I shows the

resonance Raman spectrum of the 2-pyridylhydrazone of Arthrobacter PL methylamine oxidase.

Comparisons indicate that all the amine oxidases

0031-6989/88/201V0151-2/$03.00/0

© 1988 The Italian Pharmacological Society

Pharmaco~gicalResea~h Communication~VoL2~ Supplementl~ 1988

152 contain

the

same c o f a c t o r - - P Q Q

or a similar

al. (1986); Moog et al. (1986)). spin-echo modulation

compound ( v a n

Iersel

eL

CW and pulsed EPR results (using the

technique described in McCraken et al.

(1987))

suggest that the copper sites in the bovine plasma, porcine plasma, and Arthrobacter PI enzymes are closely similar.

At least two

imidazoles and one HzO are coordinated in each case.

Another N-

containing ligand and HzO molecule may also be present.

EPR has also

been used to characterize the substrate reduced enzymes as well. Cyanide binding induces the formation of a semiquinone form that has now been characterized in considerable detail.

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I n 500000 t e n 400000 t L 300000 Y 200000

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(wavenumbers}

Resonance Raman spectrum of the 2-pyridylhydrazone of methylamine o×idase obtained with 457.9 nm excitation.

McCracken, J., Peisach, J, and Dooley, O. M. (1987)~ J. Am. Chem. Soc., [09, 4046. Moog, R., McGuirl, M., Cote, C., and Dooley, D. M. (1986), Proc. Natl. Acad. Sci. USA, 83, 8435. van lersel, J., van der Meer, R. Biochem.

, and Duine, J. (1986), Eur. J.