THE EFFECT OF HYDROGEN PEROXIDE ON PEROXIDASE (EC 1.11.1.7) FROM GONGRONEMA LATIFOLIUM

ABSTRACT

Peroxidase (EC 1.11.1.7) extracted from Gongronema latifolium was purified, on a two-step purification process of ammonium sulphate precipitation followed by dialysis. The enzyme was purified  6.8 fold with a specific activity of 2.04 when o-dianisidine was used as substrate. When the enzyme was subjected to different concentrations of hydrogen peroxide and o-dianisidine, the peak activity was 17.75µ/ml at  5mM for hydrogen peroxide and for o-dianisidine the peak activity was 2.4µ/ml observed at 0.4mM.   The optimum pH and temperature were at pH 7.0 and 30oC respectively. The Km and Vmax  for hydrogen peroxide were 1.8mM and 20u/ml and o- dianisidine had  Km of 0.12mM and Vmax of 3.3 µ/ml.  The inactivation of peroxidase extracted from Gongronema latifolium by hydrogen peroxide was time dependent and it also showed  a biphasic inactivation curve with the initial fast phase and a slower second phase. About 20% protection of the enzyme against inactivation was obtained when 1mM ascorbate was incubated in all the concentrations of   hydrogen peroxide while o-dianisidine had above 15% in all the concentrations.   Spectral studies, indicated the peak at soret band as 381 nm for the native enzyme, and when the enzyme was incubated with hydrogen peroxide, there was a shift in the soret band of the enzyme from 381nm to 389nm. Increases in the concentration of hydrogen peroxide lead to decreases in the absorbance peak at the soret band of the enzyme and also reduction of size  of Soret  band.  There were  elevations  in the  absorbance peak when1mM ascorbate and 0.4mM o-dianisidine were incubated with the enzyme at different concentrations of hydrogen peroxide

CHAPTER ONE

INTRODUCTION

The super-family of haem peroxidases from plants, fungi and bacteria is a group of enzymes that utilize hydrogen peroxide to oxidize a second (reducing) substrate often aromatic oxygen donor. These enzymes share similar catalytic cycles where hydrogen peroxide reacts with the resting ferric enzyme to form the intermediate compound I (known as compound ES in cytochrome c peroxidase) which carries two oxidizing equivalents. Compound I is subsequently reduced by reactions with two reducing substrate molecules. The reaction of these reduction steps generate the intermediate, compound II, which is then further reduced back to the ferric enzyme (Hiner et al.,2000). Peroxidase forms part of the defense system of living organisms against radical-mediated peroxidation of unsaturated lipids. They are ubiquitous in nature and are involved in various physiological processes in plants. Studies have suggested that peroxidases play a role  in  lignification, suberization, cross-linking of cell wall structural protein, auxin catabolism and self –defense against pathogens and senescence (Hiraga et al., 2001). Currently,

industrial application of peroxidase  in  chemistry,  pharmacology and  biotechnology is  well developed. Peroxidase is used in waste treatment in order to remove aromatic phenols and amine from aqueous solution in the presence of hydrogen peroxide. In this treatment, phenolic compounds are polymerized in the presence of hydrogen peroxide through a radical oxidation- reduction mechanism (Nazari et al., 2005). As hydrogen peroxide concentration increases, an irreversible mechanism-based inactivation process becomes predominant (Rodriguez-Lopez et al., 1997)  and it leads to the degradation of haem, the release of iron and the formation of two fluorescent products (Gutteridge, 1986). At a low concentration of hydrogen peroxide below

0.1mM, inactivation is predominately reversible, resulting to the formation and accumulation of catalytically  inert  intermediate  compound III.  This  inactivation of peroxidase  by  hydrogen peroxide is dependent on the concentration of hydrogen peroxide. (Zheng et al., 2001) Inactivation reaction between hydrogen peroxide and the intermediate of the enzyme’s catalytic cycle that reduced the sensitivity and efficiency of peroxidase has been studied with different sources of peroxidase, but not that of Gongronema latifolium (utazi).

1.1      PEROXIDASES

Peroxidases are known to occur in different tissues and the pattern of expression and properties of these peroxidases vary between them. Peroxidases are    haem-containing oxidoreductases (EC 1.11.1.7) that reduce peroxides, mainly hydrogen peroxide, to water and subsequently  oxidize  small  molecules,  often  aromatic  oxygen  donors  (Delannoy  et  al.,

2006).They are ubiquitous in nature and are involved in various physiological processes in plants. Studies have suggested that peroxidases play a role in lignification, suberization, cross- linking of cell wall structural proteins, auxin catabolism, self-defense against pathogens and senescence (Hiraga et al., 2001). Plant peroxidases contain two-calcium ions (Ca2+), which are essential for the structural stability and thermal stability of the enzyme as well as its in vitro activation during analysis (Manu and Prasada Rao, 2009 ; Sticher et al., 1981). Peroxidases are

widely used in clinical laboratories, industries and in environmental conservation (Lopez-Molina

et al., 2003)

1.1.1   Functional roles

Most  reactions  catalysed  by  peroxidase  especially  horseradish  peroxidase  can  be expressed by the following equation, in which AH2 and 0AH represent a reducing substrate and its radical product respectively. Typical reducing substrates include aromatic phenols, phenolic acids, indoles, amines and sulfonates.

H2O2 + 2AH2 + POD → 2H2O + 20A…………………………………………………..Reaction 1

Figure 1: Catalytic cycle of peroxidase (Villalobos and Buchanan, 2002)

During the catalytic cycle of peroxidase as shown in figure 1, the ground state enzyme undergoes a two electron oxidation by H2O2  forming an intermediate state called compound I (E). Compound I (E) will accept an aromatic compound (AH2) in its active site and will carry out

its one-electron oxidation, liberating a free radical (0AH) that is released back into the solution,

converting compound I (E) to compound II (Ei). A second aromatic compound (AH2) is accepted in the active site of compound II (Ei) and is oxidized, resulting in the release of a second free radical (0AH) and the return of the enzyme to its resting state, completing the catalytic cycle (Figure 1). The two free radicals (0AH) released into the solution combine to produce insoluble precipitate that can easily be removed by sedimentation or filtration.

Various side reactions that take place during the reaction process are responsible for the enzyme inactivation (E) or inhibition (Eii) leading to a limited lifetime, but this form is not permanent since compound III (Eii) decomposes back to the resting state of peroxidase. Some peroxidases, like horseradish peroxidase (HRP), lead to a permanent inactivation state (P-670) when H2O2  is present in excess or when the end-product polymer adheres to its active site,

causing its permanent inactivation by causing changes in its geometric configuration (Villalobos and Buchanan, 2002).

1.2.0 The structure of peroxidase

1.2.1   The description horseradish peroxidase

Horseradish peroxidase comprises a single polypeptide of 308 amino acid residue, the sequence of which was determined by Welinder, (1976). The  N-terminal residue is blocked by pyroglutamate and C-terminus is heterogeneous with some  molecules lacking terminal residue Ser308.There are four disulphide bridges between cysteine residue 11-91, 44-49, 97-301, and

177-209 and a buried bridge between Asp99 and Arg123. Nine potential N-glycosylation site can be recognized in the primary sequence from the motif Asn-X-ser/Thr (where’ X’  represents an amino acid residue) and of these, eight are occupied. A branched heptasaccharide accounts  for

75 to 80% of the glycans, but the carbohydrate profile of HRP C is heterogeneous (Yang et al.,

1996). These  invariably contain two terminal GlcNAC and several mannose residue. A further complication is the variation in the type of glycan present at any of the glycosylation site. The total carbohydrate content of the HRP C is somehow dependent on the source of the enzyme and value of between 18 and 22% typically.

Figure 2: Haem component of horseradish peroxidase isoenzyme C (HRPC) (Veitch, 2004)

I.     His170 forms coordinate bond to haem Iron

II.     Asp242 carboxylate side-chain help to control imidazolate character of His170 ring

III.      His170 Ala mutant undergoes heme degradation. When hydrogen peroxide is added and compound I and compound II are not detected, imidazole can bind to haem   Iron in the artificially created cavity but full catalytic activity is not restored because the His170 imidazole complex does not maintain a five coordinate state (His42 also binds to Fe)

IV.     Aromatic substrates are oxidized at the exposed haem edge but do not bind to haem Iron

Distal O-donors                                                                                           Proximal O- donors

Figure 3: Calcium ions component of (HRPC) (Veitch, 2004)

For the distal o-donors Asp43, Asp50, Ser52, (side chain) Asp43,Val46, Gly48 (carbonyl) one structural water. For the proximal o-donors Thr171, Asp222, Thre225, Asp230, (side chain) Thr171, Thre226, Ile228 (carbonyl)

I.     Structural water of distal calcium site hydrogen bonded to Glu64 which is itself hydrogen bonded to Asn70 and thus connect to the distal haem pocket

II.     Distal and proximal Ca2+ ions are both seven-coordinate.

III.     On calcium ions loss, enzyme activity decreases by 40%

Figure 4: Carbohydrate component of (HRPC) (Veitch,2004)

I.     Site of glycosylation are in loop regions of the structure, at Asn57, Asn13, Asn158, Asn186, Asn198, Asn214, Asn255 and Asn268.

II.     The major glycan is shown here, there are also minor glycans of the form Manm GlcNAc2

Amino acid residues

Arg38             Essential role in (i), the formation and stabilization of compound I, (ii) binding and stabilization of ligands and aromatic substrates

(e.g. benzhydroxamic acid, ferulate etc.).

Phe41               Prevent substrate access to the ferryl oxygen of compound I.

His42               Essential role in (i), compound I formation (accept proton from H2O2), (ii) binding and stabilization of ligands and aromatic substrates.

Asn70                Maintains basicity of His42 side-chain through Asn70-His42 couple (hydrogen bond from Asn70 amide oxygen to His42 imidazole NH).

Pro139             Part of a structural motif, -Pro-X- Pro- (Pro139-Ala140-Pro141 in HRP C), which is conserved in plant peroxidases

Figure 5: Key amino acid residues in the haem-binding region of HRPC. (Veitch, 2004)

HRPC contains two different types of metal centre, iron lll protoporphyrin IX (usually referred to as the haem group) and two calcium atoms.  Both are essential for the structural and functional integrity of the enzyme. The haem group is attached to the enzyme at His170 (the proximal histidine residue) by a coordinate bond between, the histidine side-chain  atom and the haem iron atom .The second axial coordination site (on the so called distal side of the haem plane) is unoccupied in the resting state of the enzyme, but available to hydrogen peroxide during enzyme turnover (Figure 5). Small molecules such as carbon II oxide, cyanide, fluoride

and azide bind to the haem iron atom at the distal site giving six-coordinate peroxidase complexes. Some bind only in their protonated forms, which are stabilized through hydrogen bonded interaction with the distal haem pocket amino  acid  side-chain of Arg38 (the distal arginine) and the His42 (the distal histidine) (Figure5). The two calcium binding sites are located at positions distal and proximal to the haem plane and are linked to the haem-binding region by a network of hydrogen bonds. Each calcium site is seven-coordinate with oxygen-donor ligands provided by a combination of amino acid side-chain carboxylate (Asp), hydroxyl group (Ser, Thr), backbone carbonyls and a structural water molecules (distal site only) asshown in figure 2 to 4. Loss of calcium results in decrease in both enzyme activity and thermal stability (Haschke and  Friedhoff, 1978) and to  subtle changes in  the haem environment that  can be detected spectroscopically (Howes et al., 2001).

.Figure 6: Three –dimensional representation of the x-ray crystal structure of HRPC (Brook haven accession code IH5A). (Veitch, 2004)

1.2.2   Three-dimensional structure of peroxidase

The first solution of the three-dimensional structure of HRP C using X-ray crystallography appeared in the literature relatively recently (Gajhede et al., 1997). The recombinant enzyme used as the source of crystals and heavy atom derivatives was produced by expression in Escherichia coli in non-glycosylated form (Smith et al., 1990). Previous attempts to obtain suitable crystals for diffraction were frustrated partly by the heterogeneity of plant HRP C preparations comprising multiple glycoforms. The structure of the enzyme is largely α-helical, although there is also a small region of β-sheet (Figure 6). There are two domains, the distal and proximal, between which the heme group is located. These domains probably originated as a result of gene duplication, a proposal supported by their common calcium binding sites and other structural elements (Welinder and Gajhede, 1993)

1.3.0 The mechanism of oxidation of peroxidase

1.3.1 Mechanisms of oxidation of indole-3-acetic acid with peroxidase

One  of the  most  interesting reactions of peroxidase (HRP-C) occurs with the plant hormone, indole-3-acetic acid (IAA) as shown in figure 7. In contrast to most peroxidase– catalysed reactions, this takes place without hydrogen peroxide, hence the use of the term ‘indole acetic acid oxidase’ to describe this activity of HRP C in the older literature. More recent studies of the reaction at neutral pH indicate that it is not an oxidase mechanism that operates, but rather a peroxidase mechanism coupled to a very efficient branched-chain process in which organic peroxide is formed (Dunford, 1999). The reaction is initiated when a trace of the indole-3-acetate cation radical is produced. The major products of indole-3-acetic acid oxidation include indole-

3-methanol, indole-3- aldehyde and 3-methylene-2-oxindole, the latter most probably as a result of the non-enzymatic conversion of indole-3-methylhydroperoxide. Conflicting theories have been proposed to explain the mechanism of reaction at lower pH ( Dunford, 1999), in   the formation  of  the  ferrous  enzyme,  compound III  and    hydroperoxyl radicals  must  also  be accounted for. The physiological significance of IAA metabolism by (HRP C) and other plant peroxidases is still an area of active debate. For example, studies on the expression of an anionic peroxidase in    transgenic tobacco  plants  indicate that  while  overproduction of the  enzyme favours defensive strategies (such as resistance to disease, physical damage and insect attack), it has a negative impact on growth due to  increased IAA degradation activity (Lagrimini, 1996).

Thus peroxidase expression in plant tissues at different stages of development must reflect a balance between the priorities of defense and growth

Figure 7: A mechanism proposed for the formation of 3-methylene-2-oxindole from horseradiperoxidase (HRP C) and indole-3-acetic acid (after Folkes et al., 2002). R represents a cellular nucleophile (e.g. sulphydryl groups of enzymes or histone)

1.3.2 Mechanism of oxidation of small phenolic substrates (Ferulic acid) with peroxidase Ferulic  acid  ((3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid.  FA)  is  a  phenolic cinnamic acid derivative that is abundant in nature and known to act as an in vivo substrate for peroxidases (Fry,1986 ). FA enhances the rigidity and strength of plant cell walls by cross- linking with pentosans,  arabinoxylans, and hemicelluloses, thereby making the cell walls less susceptible to enzymatic hydrolysis during germination. The compound is a dibasic acid that exhibits an extended resonance stabilization of the phenolate anion, hence slightly increasing its acidity relative to phenol. pKa values of 4.6 and 9.4 have been reported (Kenttamaa et al., 1970) Peroxidases have been reported to be the FA cross-linking catalyst ( Markwalder and Neukom,

1976). The level of FA and its derivatives seems to be positively correlated with protection of the plant against insects (Suga et al.,1993), fungal , viral and avian  attacks. In plants, FA is thought to arise from the conversion of cinnamic acid , and frequently it is esterified to hydroxyl groups of polysaccharides (Takahama and Oniki, 1996), flavonoids,   hydroxycarboxylic acids   and plant sterols. The initial step in the biosynthesis of lignin is the enzymatic dehydrogenation of monolignols  to produce phenoxyl radicals. The radicals can link up to form dimers, trimers, and higher oligomers.

Laccases and  plant peroxidases have been proposed to be the in vivo generators of the phenoxyl radicals. Peroxidase oxidation of compounds with a syringyl group can be enhanced by

esters of 4-coumaric acid and FA (Takahama et al.,1996). For these reasons, it is of interest to study the interactions between the cell wall component of ferulic acid  and the well characterized horseradish peroxidase C. Peroxidases catalyze the oxidative coupling of phenolic compounds using H2O2  as the oxidizing agent as shown.   The reaction is a three-step cyclic reaction by which the enzyme is first oxidized by H2O2 and then reduced in two sequential one-electron transfer steps from reducing substrates, typically a small molecule phenol derivative (the charges of heme propionates are ignored in scheme 1).

HRPC[(Fe(III))Porph2-]+ + H2O2 → HRPC[(Fe(IV)=O)Porph0-]0+ + H2O ……………Reaction 2

Native state                                                   Compound I

HRPC[(Fe(IV)=O)Porph0-]0+ + AH → HRPC[(Fe(IV)=O)Porph2-] + H+ + A0 ……….Reaction3

Compound II

HRPC[(Fe(IV)=O)Porph2-] + H+   + AH  → HRPC[(Fe(III))Porph2-]+  + H2O + A0………Reaction4

SCHEME 1: Reactions 2–4

The oxidized phenolic radicals polymerize with the final product depending on the chemical character of the radical, the environment, and the peroxidase isoenzyme used (Frias et al.,1991). The oxidation of native enzyme by H2O2 is well understood, and numerous experiments  have confirmed the general catalytic mechanism for this step first proposed by (Poulos and Kraut,

1980). The oxidation of phenolic substrates (reactions 4 and 4) is less well understood, but a histidine  (His42 in HRPC) and an arginine  (Arg38 in HRPC) (Rodriguez-lopez et al., 1997) have been shown to contribute significantly to enhance the rate of substrate oxidation.

Figure 8: Proposed mechanism for substrate oxidation in plant peroxidases. (Poulos and Kraut,

1980)

First, the active site arginine (Arg38 in HRPC) donates a hydrogen bond to the phenolic oxygen of the reducing substrate. This hydrogen bond  will assist  proton transfer from the phenolic oxygen to active site histidine (His42 in HRPC) through an active site water molecule held in position by the backbone oxygen of a conserved proline residue (Pro139 in HRPC). The electron is transferred to the haem group via the C-18 methyl-C-20 haem edge. Then compound II reduction is assisted by a similar proton transfer. The proton can be transferred to the ferryl oxygen through the active site water molecule situated equidistant between the distal histidine and the expected position of the ferryl oxygen of compound II, regenerating the resting state enzyme and a water molecule (Henriksen et al., 1999)

1.4      Classes of peroxidases

Peroxidases, a class of enzymes in animals, plants and microorganisms, catalyze oxidoreduction between H2O2  and various reductants. Peroxidases fall into two major super families according to their primary sequence: animal and plant peroxidases (Table 1).

Table 1: Classification of peroxidases (Hiraga et al., 2001)

CLASSES

(EC NUMBER) MEMBER

ORIGIN                    MOLECULAR

  SUPERFAMILY         (PEROXIDASE)                                                        WEIGHT(KDA)         

Animal peroxidase       Eosinophill         peroxidase

(EC1.11.1.7)

Lacto                  peroxidase

(EC1.11.1.7)

Myclo                 peroxidase

(EC1.11.1.7)

Thyroid              peroxidase

(EC1,11.1.9)

Glutathione        peroxidase

(EC1.11.1.7)

Prostaglandin endoperoxidase (EC1.14.99.1,partial)

Animal                       50-75

Animal                       78-85

Animal                       79-150

Animal                       90-110

Animal                       6-22 and 75-112b

Animal                       115-140

Catalase                        Catalase (EC 1.11.1.6)            Animal				Plant	140-530
Fungus and Yeast
Plant Peroxidase	Cytochrome  C	peroxidase	Bacterium	and	32-63
	(EC 1.11.1.6) Catalase	peroxidase	Yeast Bacterium	and	150-240
	(EC1.11.1.6)		Fungus
	Ascorbate (EC1.11.1.11)	peroxidase	Plant		30-58

Manganese-dependent peroxidase (EC1.11.1.13)

Fungus                       43-49

Ligninase (EC1.11.1.14)         Fungus                       40-43

Peroxidase(EC1.11.17POX)   Plant                          28-60

1. 5.    Plant peroxidase

1.5 .1 Plant peroxidases

Based on differences in primary structure, the plant peroxidase super family can be further divided into three classes (Table 1).  The plant peroxidases, which share similar overall protein folds and specific features, (such as catalytically essential histidine and arginine residues in their active sites), have been subdivided into three classes on the basis of sequence comparison (Welinder, 1991). In class I are intracellular enzymes including yeast cytochrome c peroxidase, ascorbate peroxidase (APX)  from plants, and bacterial gene duplicated catalase-peroxidases (Welinder,1991). Class II consists of the secretory fungal peroxidases such as lignin peroxidase (LiP) from Phanerochaete chrysosporium, manganese peroxidase from the same source, and Coprinus cinereus peroxidase or Arthromyces ramosus peroxidase (ARP), which have been shown to be essentially identical in both sequence and properties (Kjalke et al., 1992). The main role of class II peroxidases appears to be the degradation of lignin in wood. Class III contains the secretory plant peroxidases such as those from horseradish (HRP), barley and soybean. These peroxidases seem to be biosynthetic enzymes involved in processes such as plant cell wall formation, and lignifications as shown in (Figure 9)

1.5.2 Functions of plant peroxidase

Plant  peroxidases  have  often  been  suggested  to  be  involved  in  the  biosynthesis  of complex cell wall macromolecules such as lignin and suberin, both of which are synthesized by plant for mechanical strength, defense, restoring damaged tissues, and water transport (Vidali,

2001 and De Gara, 2004). Plant peroxidases (PODs) oxidise phenolic domains of feruloylated polysaccharides and tyrosine residues of cell wall structural proteins such as hydroxyproline-rich glycoproteins to form more complex and larger molecules in the cell wall, thereby restricting cell expansion and pathogen invasion. In tobacco, a positive correlation was found between PODs activity and  resistance to tobacco wildfire disease. The roles of PODs in defense are considered as follows:

I.      Reinforcement of cell wall physical barriers comprising lignin, suberin, feruloylated polysaccharides and hydroxyproline-rich glycoproteins.

II.      Enhancement   of   reactive   oxygen   species   production   as   signal   mediators   and antimicrobial agents.

III.      Enhancement of phytoalexin production.

Generally, multiple PODs are induced by pathogen infection, suggesting that each POD is involved in a specific defense process (Hiraga et al., 2001 and Cosio and Dunand, 2009). Peroxidases from tobacco  and  HRP have  showned  higher specific  activities  to  NADH, NADPH and IAA than to  monolignols, suggesting their  involvement in some cell wall biosynthetic processes other than polymerization of monolignols (Figure 9) (Delannoy et al.,

2006 ; Cosio and Dunand, 2009).

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