Báo cáo khoa học: Active site residue involvement in monoamine or diamine oxidation catalysed by pea

Active site residue involvement in monoamine or diamine

oxidation catalysed by pea seedling amine oxidase

Maria Luisa Di Paolo1

, Michele Lunelli2

, Monika Fuxreiter2,3, Adelio Rigo1

, Istvan Simon3

and Marina Scarpa2

1 Dipartimento di Chimica Biologica and INBB, Universita` di Padova, Padova, Italy

2 Dipartimento di Fisica, Universita` di Trento, Trento, Italy

3 Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary

Introduction

Copper amine oxidases (CuAOs; EC 1.4.3.6) are wide￾spread in nature, being present in both prokaryotic

and eukaryotic organisms. They are homodimers, each

subunit containing a copper and a redox cofactor,

2,4,5-trihydroxyphenylalanine quinone (TPQ) [1].

CuAOs catalyse the oxidative deamination of primary

amines to the corresponding aldehydes, according to

the overall reaction:

RCH2NHþ

3 þ O2 þ H2O ! RCHO þ NHþ

4 þ H2O2

Catalysis occurs by a ping-pong mechanism, in which

the amine is converted to the product aldehyde while

reducing the enzyme cofactor (reductive half-reaction);

this is followed by reoxidation of the cofactor by

oxygen, which completes the catalytic cycle (oxidative

half-reaction) [2].

To date, several amine oxidase crystal structures

have been solved [3–10]. The structures for Escherichia

coli (ECAO) [3], Pisum sativum (PSAO) [4], Arthro￾bacter globiformis (AGAO) [5], Hansenula polimorpha

(HPAO) [6], Pichia pastoris [7], bovine serum amine

oxidase (BSAO) [8] and human semicarbazide sensitive

amine oxidase [9,10] reveal the similarity of the overall

fold of these enzymes from various sources and point

to the importance of the channel involved in amine

substrate binding. The domain including the catalytic

region (called D4) exhibits a rather high sequence

similarity. All these features may implicate a similar

Keywords

amine oxidase; substrate docking; substrate

selectivity; substrate-dependent catalytic

mechanism; titratable amino acids

Correspondence

M. Scarpa, Dipartimento di Fisica, Via

Sommarive 14, 38050 Povo-Trento, Italy

Fax: ++39 0461881696

Tel: ++39 0461882029

E-mail: [email protected]

(Received 13 October 2010, revised 24

December 2010, accepted 2 February 2011)

doi:10.1111/j.1742-4658.2011.08044.x

The structures of copper amine oxidases from various sources show good

similarity, suggesting similar catalytic mechanisms for all members of this

enzyme family. However, the optimal substrates for each member differ,

depending on the source of the enzyme and its location. The structural fac￾tors underlying substrate selectivity still remain to be discovered. With this

in view, we examined the kinetic behaviour of pea seedling amine oxidase

with cadaverine and hexylamine, the first bearing two, and the second only

one, positively charged amino group. The dependence of Km and catalytic

constant (kc) values on pH, ionic strength and temperature indicates that

binding of the monoamine is driven by hydrophobic interactions. Instead,

binding of the diamine is strongly facilitated by electrostatic factors, con￾trolled by polar side-chains and two titratable residues present in the active

site. The position of the docked substrate is also essential for the participa￾tion of titratable amino acid residues in the following catalytic steps. A

new mechanistic model explaining the substrate-dependent kinetics of the

reaction is discussed.

Abbreviations

AGAO, Arthrobacter globiformis amine oxidase; BSAO, bovine serum amine oxidase; CAD, cadaverine; CuAO, copper amine oxidase; ECAO,

Escherichia coli amine oxidase; HEX, hexylamine; HPAO, Hansenula polimorpha amine oxidase; I, ionic strength;) kc, catalytic constant;

PSAO, Pisum sativum amine oxidase; T, temperature; TPQ, 2,4,5-trihydroxyphenylalanine quinone.

1232 FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS