Substrate specificity of copper-containing plant amine oxidases

Dedicated to the memory of Olivia Befani, prematurely deceased on February, 2006.
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Abstract

The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The kcat and kcat/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of kcat versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pKa of both enzyme-substrate and enzyme-product adducts. The plot of kcat/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pKa couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pKa. The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.

Introduction

Copper-containing amine oxidases (AOs) are widespread in nature, occurring in both prokaryotic and eukaryotic organisms [1]. They catalyze the oxidative deamination of primary amines to aldehydes with a ping-pong mechanism consisting of a transamination [2], followed by the transfer of two electrons to molecular oxygen which is reduced to H2O2 (Eqs. (1), (2)).Eox+RCH2NH3+EredNH3++RCHOEredNH3++O2Eox+NH4++H2O2

In addition to copper, they contain an organic prosthetic group reactive with semicarbazide, phenylhydrazine, and similar inhibitors of the catalytic activity. This was identified [3] as tri-hydroxyphenylalanine quinone (TPQ), a posttranslationally oxidized tyrosine residue [4]. The role of amine oxidases is not always well defined. The metabolism of primary amines provides a source of carbon and nitrogen for bacterial growth. In plants, the production of hydrogen peroxide deriving from polyamine oxidation has been correlated with cell wall maturation and lignification during development as well as with wound-healing and cell wall reinforcement during pathogen invasion [5]. In higher organisms they have been implicated as key components in processes such as leukocyte trafficking involving the CuAO vascular adhesion protein-1 (VAP-1) [6].

The X-ray structure of several amine oxidases showed that the enzymes are dimeric, with extensively interacting subunits of 70–90 kDa and considerable structural homology [7], [8], [9], [10], [11], [12], [13], [14]. Thirty-three residues in the catalytic site region are fully conserved in 10 enzymes from different sources [15], although the overall primary sequence identity is usually not high, <25%. A high degree of identity was reported for the bovine serum amine oxidase (BSAO) and VAP-1 pair (83%) [12] and the pea and lentil seedling amine oxidases (PSAO and LSAO, respectively) pair (91%) [16]. Other plant amine oxidases probably share this property since the N-terminal 30 amino acid sequences of Lathyrus odoratus and Lathyrus sativus amine oxidase show 100% identity with PSAO [17].

The structural similarity does not prevent amine oxidases of various sources from displaying different reactivity with substrates and inhibitors [2], [18]. An example is provided by LSAO and the amine oxidase from bovine serum (BSAO). LSAO is a quite active diamine oxidase, and produces the semiquinolamine–Cu+ radical in equilibrium with quinolamine–Cu2+ upon reduction by substrates under anaerobic conditions [19]. BSAO has an extremely poor reactivity with putrescine [20], and does not form the radical, under experimental conditions in which LSAO does [18]. The reasons for the differences are not clear. An influence on the reactivity was proposed for the size of the hydrophobic channel leading to the active site, deeply buried inside the protein molecule, since the channel is wider in PPLO [11] and BSAO [12] than in other amine oxidases.

In the attempt to clarify these points, an investigation was undertaken on the catalytic parameters of the plant enzymes with a set of substrates similar to that previously used with BSAO [20]. The comparison of the data, together with available information on the X-ray structure of the catalytic sites, may provide an explanation of some observed differences. The enzymes would be used as natural site-specific mutants, since site-specific mutations were never reported for mammalian and plant amine oxidases. The two amine oxidases from plant examined in the present work were purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling. Unfortunately, the X-ray structure of PSAO [8] shows TPQ in a conformation unsuited to substrate binding. A ∼180° rotation of the TPQ plane about the Cβ–Cγ bond was suggested to precede the binding in order to make the C5 carbonyl available to attack by the substrate amino group entering through the hydrophobic channel and to H+ abstraction by Asp300 [8]. For LCAO not even the primary structure is known, but the protein shows substrate specificity quite different from that of BSAO [21]. The interest in the properties of these proteins was raised on one side by BSAO similarity with human VAP-1 and on the other side by recent reports on their possible therapeutic use, related to their greater ability than BSAO to metabolize histamine, which is the principal mediator in the first phase of allergic reactions in mammals [22] and is involved in other pathologies like heart ischemia [23] and ulcerative colitis [24].

Section snippets

Protein purification and characterization

All chemicals were reagent grade and were used without further purification. Substrates and peroxidase were purchased from Sigma–Aldrich S.r.l. (Milan, Italy). LCAO and PSAO were purified from L. cicera seedling and from pea seedling by the method previously reported [21]. The purified proteins moved as single bands on SDS–PAGE. The concentration was measured by employing for both enzymes the molar extinction coefficients reported for PSAO [21], [25], namely ε280nm = 300 mM−1 cm−1 and ε500nm = 4.9 mM−1

Substrate specificity

Fig. 1 shows the substrates examined in the present work. The steady-state kinetic parameters for their oxidation catalyzed by LCAO are reported in Table 1. Some of the pH 7.2 parameters were reported in a previous work [21]. They show small differences, which might be imputed to differences in ionic strength, that was kept constant at 120 mM in present experiments. Some substrates were also tested on PSAO with very similar results. Since PSAO preferred pH values are known to be pH 7.2 and pH

Conclusions

The results of the present investigation indicate major differences of reactivity between plant amine oxidases and BSAO and suggest a possible explanation for some differences. In agreement with Cogoni et al. [38], LCAO was found to be a diamine oxidase as PSAO, having a kcat for diamines, putrescine in particular, much higher than that of BSAO. However, the turnover was considerably higher than that of BSAO also with other substrates such as histamine and some aromatic amines (tyramine and

Abbreviations

    AO

    amine oxidase

    AGAO

    Arthrobacter globiformis amine oxidase

    BSAO

    bovine serum amine oxidase

    ECAO

    Escherichia coli amine oxidase

    HPAO

    Hansenula polymorpha amine oxidase

    2HP

    2-hydrazinopyridine

    LCAO

    Lathyrus cicera amine oxidase

    LSAO

    lentil seedling amine oxidase

    PPLO

    Pichia pastoris lysyl oxidase

    PSAO

    pea seedling amine oxidase

    TPQ

    2,4,5-trihydroxyphenylalanine quinone

    VAP-1

    vascular adhesion protein-1

Acknowledgments

We gratefully acknowledge the helpful discussion and suggestions of Veronica Morea and Anna Tramontano.

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