Biochem. J. (1970) 117, 169-176 169Printed in Great Britain

A Reinvestigation of the Substrate Specificity of Pig Kidney DiamineOxidase

BY W. G. BARDSLEYDepartment of Biochemistr?y, University of Manchester Institute of Science and Technology,

Manchester M60 1QD, U.K.

AND C. M. HILLDepartment of Physiology, University of Manchester, Manchester M1i13 9PL, U.K.

AND R. W. LOBLEY*Departmnent of Chemistry and Applied Chemistry, University of Salford, Salford M5 41VT, U.K.

(Received 4 November 1969)

1. The substrate specificity of pig kidney diamine oxidase was reinvestigatedwith a purer enzyme preparation than has previously been used for this purpose.2. All substrates were extensively purified before use, and methods of preparationor sources are given, together with RF values. 3. The substrate specificity deter-mined differed somewhat from that reported by previous workers and, in addition,the behaviour of several compouinds not previously used as substrates is described.4. A model for enzyme-substrate interaction embodying these observations isformulated. It is suggested that a negatively charged substrate-binding group issituated at 6.0-9.0A from the oxidizing site. The binding and oxidizing sites areseparated by a hydrophobic or methylene-binding site.

The enzyme known as pig kidney diamineoxidase or histaminase [diamine-oxygen oxido-reductase (deaminating), EC 1.4.3.6] was first re-ported by McHenry & Gavin (1932), and the sub-strate specificity was extensively investigated byZeller, Fouts, Carbon, Lazanas & Voegtli (1956),Fouts, Blanksma, Carbon & Zeller (1957), Blaschko& Chrusciel (1959) and Zeller (1963). The enzymehas since been obtained in a homogeneous form(Mondovi et al. 1967) and crystallized (Yamada,Kumagai, Kawasaki, Matsuii & Ogata, 1967).

It is now known to have a molecular weight of185 000, to contain Cu2+ ions, which do not change invalency as oxidation proceeds, and to containpyridoxal phosphate, which is involved in substratebinding (Kumagai, Nagate, Yamada & Fukami,1969). The enzyme catalyses the oxidation ofdiamines thus (Tabor, 1951):

H2N* [CH2]1 .NH2 + 02+ H20=H2N * [CH2]n1 CHO + H202 +NH3

and also weakly catalyses the oxidation of somemonoamines. The mode of enzyme-substrate inter-action is ustually depicted as in Scheme 1, where anoxidizing site binds one amino group as a cation,

* Present address: Department of Gastroenterology,Manchester Royal Infirmary, Manchester M13 9WL, U.K.

and where the other amino group is uncharged andacts as a nucleophile towards an electron-acceptingsite, which is suggested to be a carbonyl group.However, since this scheme does not fully explain

the substrate pattern, we now describe a new modelfor substrate binding, based on a reinvestigation ofthe substrate specificity, which more adequatelyaccouints for the known behaviour of this enzyme.

MATERIALS AND METHODS

Table 1 lists all amines and amino aci(ls used in thepresent study together with sources.

-NH2IH

-, EA PA-

Scheme 1. Interaction between a diamine and diamineoxidase (after Zeller, 1956). One amino group is un-charged and acts as a nucleophile towards an electron-accepting site (EA, which is suggested to be a carbonylgroup). The amino group to be oxidized is protonated andis attracted to a proton-accepting group (PA-), which isnegatively charged. Zeller (1951) originially sucggested thatboth sites were negatively chtarge(l.

1970W. G. BARDSLEY, C. M. HILL AND R. W. LOBLEYTable 1. Oxidation of amine8 and amino acids by pig kidney diamine oxida8e

The rate of oxidation was determined manometrically with a final substrate concentration of 3.3mM, andis presented as a percentage ofthe oxidation of cadaverine. A comparison with literature values is given wherepossible, taken from Zeller (1963), Zeller et al. (1956), Fouts et al. (1957) and Blaschko & Chrus6iel (1959). Herethe conditions were varied and precise values not always given. + Denotes a small but definite 02 uptake.Inter-nitrogen-atom distances in diamines were measured by using Prentice-Hall framework molecularmodels. All compounds used were purified until only one spot was observed on t.l.c. and RF values refer to oneof three systems: A, propan-2-ol-aq.NH3 (sp.gr.0.88)-water (10:1:1, by vol.); B, methanol-aq.NH3 (sp.gr.0.88)-water (10:2:1, by vol.); C, acetic acid-water (10:1, v/v). Sources of compounds were as follows: A,Aldrich Chemical Co. Inc., Milwaukee, Wis., U.S.A.; BDH, BDH Chemicals Ltd., Poole, Dorset, U.K.; KK,K & K Rare Chemicals, Plainview, N.Y., U.S.A.; KL, Koch-Light Laboratories Ltd., Colnbrook, Bucks.,U.K.; P, prepared by us (see the Materials and Methods section); RNE, R. N. Emanuel Ltd., Alperton, Middx.,U.K.; S, Sigma (London) Chemical Co., London S.W.6, U.K.

Compound1,2-Diaminoethane dihydrochloride1,3-Diaminopropane1,4-Diaminobutane (putrescine)1,5-Diaminopentane dihydrochloride(cadaverine dihydrochloride)

1,6-Diaminohexane1,7-Diaminoheptane1 ,8-Diamino-octaneci8-1,4-Bis(aminomethyl)cyclohexaneo-Bis(aminomethyl)benzene(o-xylylenediamine)

m-Bis(aminomethyl)benzene(m-xylylenediamine)

p-Bis(aminomethyl)benzene(p-xylylenediamine)

o-Bis-(2-aminoethyl)benzenem-Bis-(2-aminoethyl)benzenep-Bis-(2-aminoethyl)benzene3-Dimethylaminoprop-1-ylamine4-Dimethylaminobut-1-ylamine5-Dimethylaminopent-1-ylamine6-Dimethylaminohex-1-ylamineo-Bis-(N-methylaminomethyl)benzenem-Bis-(N-methylaminomethyl)benzenep-Bis-(N-methylaminomethyl)benzeneo-Bis-(NN-dimethylaminomethyl)benzenem-Bis-(NN-dimethylaminomethyl)benzenep-Bis-(NN-dimethylaminomethyl)benzeneI-Aminopropane1-Aminobutane1-AminopentaneBenzylaminePhenethylamine3-Phenylprop-1-ylamine4-Phenylbut-1-ylamineHistamine dihydrochlorideTryptamine hydrochlorideTyramine hydrochlorideNoradrenaline bitartrateMescaline sulphate3-Aminopropionic acid4-Aminobutyric acid5-Aminovaleric acid6-Aminohexanoic acid (6-aminocaproic acid)o-Aminomethylbenzoic acidm-Aminomethylbenzoic acidp-Aminomethylbenzoic acid

SourceBDHBDHKLKL

BDHKLKLAp

A

p

pppKLKKKKKKppppppBDHBDHBDHBDHBDHpKKS

KLKLKLKLKLKLRNEKT.ppp

Rate of oxidation asa percentage of

cadaverine oxidationRF value.,

andsolvent0.25,B0.19, B0.08, B0.06, B

0.10,B0.10,B0.14,B0.14,B0.33, A

0.36,A

0.36, A

0.20, A0.27, A0.35, A0.22, B0.13, B0.14,B0.19, B0.51, A0.52,A0.34,A0.75, A0.44, A0.89, A0.58,00.63,00.68,00.71, B0.65, B0.50, B0.54, B0.52, B0.74, A0.68, B0.58,00.65, B0.41, B0.47 ,B0.43, B0.49, B0.55, B0.65, B0.71, B

Literaturevalue52595100

555515

Presentwork

48

96100

41431600

0 +

0 48

_0

7+

550 3255 28

80

0

0

0

0

0

25 710 9+ +

+ 0

+

0

0

50 51_ O+ 0

0

10 00

0

0

0

0

0

0

Inter-nitrogen-atom distancefor diamines

(extreme valuespossible in

parentheses) (A)4.1 (2.7-4.1)5.5 (3.1-5.5)6.5 (3.0-6.5)7.0 (3.0-8.0)

8.1 (3.0-9.2)9.0 (3.0-10.5)

10.5 (3.0-12.0)8.0 (4.3-8.0)6.3 (3.0-6.3)

7.2 (5.1-7.7)

8.0 (7.3-8.1)

7.9 (3.0-8.1)9.6 (3.0-10.5)

10.4 (7.3-11.3)5.5 (3.1-5.5)6.5 (3.0-6.5)7.0 (3.0-8.0)8.1 (3.0-9.2)6.3 (3.0-6.3)7.2 (5.1-7.7)8.0 (7.3-8.1)6.3 (3.0-6.3)7.2 (5.1-7.7)8.0 (7.3-8.1)

6.0 (3.5-6.4)

170

Number(I)(II)(III)(IV)

(V)(VI)(VII)(VIII)(IX)

(X)

(XI)

(XII)(XIII)(XIV)(XV)(XVI)(XVII)(XVIII)(XIX)(XX)(XXI)(XXII)(XXIII)(XXIV)(XXV)(XXVI)(XXVII)(XXVIII)(XXIX)(XXX)(XXXI)(XXXII)(XXXIII)(XXXIV)(XXXV)(XXXVI)(XXXVII)(XXXVIII)(XXXIX)(XL)(XLI)(XLII)(XLIII)

171SUBSTRATE SPECIFICITY OF DIAMINE OXIDASE

Table 2. Summary of the preparation of pig kidney diamine oxidase

Step1. Homogenate2. Thermal denaturation3. Ammonium sulphate

fractionation I4. Column chromatography on

hydroxyapatite5. Ammonium sulphate

fractionation II6. Column chromatography on

DEAE-cellulose

Total volume Total protein(ml) (mg)1650 2490001440 26900210 4700

125

6

6

Total activity(units)11073.871.1

874 59.3

268 38.4

68 18.5

Specific activity(unit/mg)0.00040.00270.0151

0.0679

0.143

0.274

Twice-recrystallized catalase was obtained from Sigma(London) Chemical Co., London S.W.6, U.K., DEAE-cellulose (type DE32) was from W. and R. Balston Ltd.,Maidstone, Kent, U.K., DEAE-Sephadex and SephadexG-200 were from Pharmacia (G.B.) Ltd., London W.13,U.K., semicarbazide hydrochloride, hydrazine hydro-chloride, hydroxyammonium sulphate, sodium diethyl-dithiocarbamate, methylammonium chloride, trimethyl-ammonium chloride and tetramethylammonium chloridewere purchased from BDH Chemicals Ltd., Poole, Dorset,U.K. Adsorbent used for t.l.c. was Kieselgel GF254 (E.Merck A.-G., Darmstadt, Germany).

Mea8urement of substrate oxidation. Oxygen uptake wasmeasured by conventional Warburg manometry at 37°Cwith air as the gas phase. A final volume of 3ml of 0.1M-

potassium phosphate buffer, pH 7.0, was used, containingenzyme (approx. 0.1unit), catalase (46,ug) and a finalsubstrate concentration of 3.3mM.One unit of diamine oxidase is defined as the amount of

enzyme that catalyses the transformation of l,umol ofsubstrate/min under the above standard conditions, andis equivalent to an oxygen uptake of 11.2,ul/min.

Protein determination. Protein was estimated spectro-photometrically from the E280 value in 0.1M-potassiumphosphate buffer, pH 7.0. An extinction coefficient(EBO-"°) of 1.63 was assumed (Yamada et al. 1967).Enzyme preparation. All steps except 1 and 2 were car-

ried out at 0-40C and all buffers were saturated with tolu-ene. The enzyme preparation is summarized in Table 2.

Step 1. Fresh pig kidney cortex (1.5 kg) was homogen.ized in a top-drive macerator (Townson and Mercer) in0.1M-potassium phosphate buffer, pH7.0 (1200ml), andcentrifuged.

Step 2. The supernatant was heated at 60°C for 10min,cooled and centrifuged.

Step 3. The supernatant was fractionated with am-

monium sulphate. The precipitate formed between 1.2M-and 2.5M-ammonium sulphate was collected and dialysedagainst 0.1m-potassium phosphate buffer, pH7.0.

Step 4. The dialysis residue was applied to a hydroxy-apatite column (4.5 cm x14.5cm) and washed with 0.1M-

potassium phosphate buffer, pH 7.0, containing 0.2m-ammonium sulphate. The enzyme was eluted by raisingthe ammonium sulphate concentration to 0.5m.

Step 5. Active fractions were combined, and the pre-cipitate formed between 1.4M- and 2.4M-ammonium

sulphate was collected, dialysed against 0.05 M-potassiumphosphate buffer, pH 7.2, and concentrated to a finalvolume of 6 ml.

Step 6. The concentrate was dialysed (0.05M-potassiumphosphate buffer, pH7.2) and applied to a DEAE-cellulose column (2.5cmx21cm). Gradient elution was

carried out by using a Varigrad device containing 0.05M-potassium phosphate buffer, pH 7.2 (150ml), in compart.ment 1, 0-2 M-potassium phosphate buffer, pH 7-2(150ml), in compartment 2 and 0.5m-potassium phos-phate buffer, pH7.2 (150ml), in the third compartment.The enzyme was precipitated from the pooled active frac-tions with ammonium sulphate (2.4m,), dissolved in O.1M-potassium phosphate buffer, pH 7.0 (6 ml), dialysed againstthe same buffer, centrifuged and stored at 4°C. Afterstorage for 6 months, the activity was half the originalvalue.

Measurement of inter-nitrogen distances in diamines. Anapproximate measure of the distance between the centresof the two nitrogen atoms in diamine cations was obtainedby using molecular models (Prentice-Hall frameworkmolecular models). Table 1 lists estimations made fromthe most probable conformations adopted in solution,allowing for repulsion between the centres of positivecharge and minimizing eclipsed conformations of methy.lene chains. The values quoted are obviously more

accurate for aromatic and short-chain aliphatic diaminesthan for longer-chain aliphatic diamines.

Preparation of hydrochlorides. All compounds employedas potential substrates in this study, except compounds(XXXV)-(XLIII) inclusive, were used as hydrochlorides.These were prepared by cautious addition of a slightexcess of conc. HCI to an ethanolic solution of the amine,followed by evaporation to dryness under reducedpressure. The residues were then repeatedly recrystal-lized until seen to be homogeneous on t.l.c. and pure as

judged by melting points.Melting points. A Kofler block was used and the values

are uncorrected.Spectroscopic measurement8. All compounds prepared

were identified unambiguously by i.r. and n.m.r. spectro.scopy. In addition, new compounds were further identi-fied by mass spectrometry and elemental analysis ofderivatives.

Preparation of amines and amino acids. o-Bis(amino-methyl)benzene (IX). o-Bis(phthalimidomethyl)benzene

Vol. 117

Yield(%)1006765

Purification16

34

53 154

35 323

17 634

W. G. BARDSLEY, C. M. HILL AND R. W. LOBLEY

(3g), prepared by the method of Strassmann (1888), wasrefluxed in a solution of 100% hydrazine hydrate (2ml),ethanol (25ml) and ehlorobenzene for 3h. o-Bis(amino-methyl)benzene was then extracted with acid, andobtained as a white crystalline hydrochloride (1.14g,72% yield).p-Bis(aminomethyl)benzene (XI). This was prepared

by a similar method from p-bis(bromomethyl)benzene,and obtained as a hydrochloride, m.p.>3600C (Found: C,46.0; H, 6.8; N, 13.4; C8H14Cl2N2 requires C, 46.0; H, 6.7;N, 13.4%).

o - Bis - (2 - aminoethyl)benzene (XII). o - Bis(cyano-methyl)benzene (3.9g) was reduced with H2 at atmos-pheric pressure and at 60°C in acetic anhydride(75ml) by using Adams platinum catalyst (0.4g). The coolfiltered solution was evaporated to give o-bis(acetamido-methyl)benzene (5.87g, 95% yield). A recrystallizedsample of this (2.1g) was hydrolysed with refluxingmethanolic KOH (25% KOH in methanol) to give o-bis-(2-aminoethyl)benzene (1.24g, 87% yield), b.p. 88-90°C/0.15mmHg.

m-Bis-(2-aminoethyl)benzene (XIII). This was pre-pared by the method of Ruggli & Prijs (1945) and hadb.p.104-108'C/0.2mmHg.p-Bis-(2-aminoethyl)benzene (XIV). This was pre-

pared by the method of Ruggli & Prijs (1945) and hadb.p.104-106°C/0.15mmHg.o-Bis-(N-methylaminomethyl)benzene (XIX). A mix.

ture of NN'-dimethylhydrazine dihydrochloride (10g),dimethylformamide (20ml) and anhydrous K2CO3 (20g)was agitated at 1000C until no further CO2 was released.o-Bis(bromomethyl)benzene (lOg) was added and thesolution refluxed for 20min. After filtration and acidifica-tion, the solution was evaporated to dryness under reducedpressure and the basified residue extracted with ether togive a clear oil, which on distillation gave 1,2-dimethyl-4,5-benzo-1,2,3,6-tetrahydropyridazine, b.p. 64-66°C/0.05mmHg (3.7g, 30.2% yield). The n.m.r. spectrum inCdC03, with tetramethylsilane as the internal reference,showed six N-methyl protons (7.6T, singlet), four ring-methylene protons (6.15T, singlet) and four aromatic pro-tons (2.85-, multiplet). Junge & Staab (1967) quoteb.p. 63-65°C/0.OOlmmHg and ring-methylene protons(6.19 r, singlet) for this compound.A sample of this distillate (3.12g) was reduced with zinc

powder (30g) and 5M-HCI (150ml) for 2h at 1000C to giveo-bis-(N-methylaminomethyl)benzene [3.2g, quantitativeyield; the n.m.r. spectrum in trifluorodeuteroacetic acid,with tetramethylsilane as internal standard, showed sixN-methyl protons at 6.965 (triplet), four methyleneprotons at 5.36r (triplet) and four aromatic protons at2.32r (singlet)]. The material was purified by conversioninto the dipicrate, m.p. 182-185°C (Found: C, 42.5; H, 3.9;N, 18.4; C22H22N8014 requires C, 42.4; H, 3.6; N, 18%),which was recrystallized and reconverted into the freebase (overall yield 80%).The dihydrochloride of o-bis-(N-methylaminomethyl)-

benzene had m.p. 263-264°C in a sealed tube (Found: C,50.8; H, 7.6; Cl, 30.2; N, 11.7; C10H18C12N requires C,50.6; H, 8.0; Cl, 29.9; N, 11.8%).

m-Bis-(N-methylaminomethyl)benzene (XX). Thiswas prepared by the method of Sander & Burmeister(1962), and had b.p.120-122°C/4.5mmHg. The hydro-chloride was hygroscopic, and had m.p.132-] 3400 (sealed

tube) (Found: C, 50.7; H, 8.0; C1, 29.7; N, 11.7;C0oHj8C12N2 requires C, 50.6; H, 8.0; Cl, 29.9; N, 11.8%).p-Bis-(N-methylaminomethyl)benzene (XXI). A mix-

ture of p-bis(bromomethyl)benzene (lOg) and ethanolic30% methylamine (lOOml) was maintained at 7000 for 4 hin an autoclave. After removal of solvent, basic materialwas extracted with acid and distilled to give p-bis-(N-methylaminomethyl)benzene, b.p. 110-112°C/4.5mmHg(1.3g, 22% yield). The hydrochloride had m.p.293-295°Cin a sealed tube (Found: C, 50.6; H, 7.6; Cl, 30.5; N, 11.9;C0oH18C12N2 requires C, 50.6; H, 8.0; Cl, 29.9; N, 11.8%).

o-Bis-(NN-dimethylaminomethyl)benzene (XXII). Themethod of von Braun & Cahn (1924) for preparing thiscompound gave, in our hands, low yields of impurematerial, and also proved hazardous. A simple modifica-tion is now described. o-Bis-(NN-dimethyl-N-phenethyl-aminomethyl)benzene dibromide (18g), prepared asdescribed by von Braun & Cahn (1924), was suspended in4M-NaOH (200ml). After being stirred for 45min at1000C the cooled solution was partitioned between 3M-HCI and chloroform. The acid layer was then basified, andextracted with chloroform to yield a clear oil, which ondistillation gave o-bis-(NN-dimethylaminomethyl)ben-zene, b.p.64-680C/0.2mmHg (5.8g, 95% yield). Thehydrochloride was hygroscopic and had m.p.216-218°C(sealed tube) (Found: C, 54.5; H, 8.4; C1, 26.9; N, 10.3;C12H22C12N2 requires C, 54.5; H, 8.4; Cl, 26.8; N, 10.6%).

m-Bi8-(NN-dimethylaminomethyl)benzene (XXIII). Thiswas prepared as described for compound (XXI) fromm-bis(bromomethyl)benzene and dimethylamine in 66%yield, and had b.p.54-560C/O.lmmHg. The n.m.r.spectrum in carbon tetrachloride, with tetramethylsilaneas internal reference, showed twelve N-methyl protons(7.84r, singlet), four methylene protons (6.64-i, singlet)and four aromatic protons (2.87T, multiplet). The dipi-crate had m.p.205-206'C (Found: C, 44.2; H, 4.1; N,17.5; C24H26N,8014 requires C, 44.3; H, 4.0; N, 17.2%).The hydrochloride was hygroscopic, and had m.p.150-

1520C (sealed tube) (Found: C, 54.1; H, 8.3; C1, 26.7; N,10.6; C12H22C02N2 requires C, 54.4; H, 8.4; Cl, 26.8; N,10.6%).p-Bis-(NN-dimethylaminomethyl)benzene (XXIV).

This was prepared by the method of Fusco, Chiavarelli,Palazzo & Bovet (1948), and had b.p.94-960C/0.4mmHg.

3-Phenylprop-1-ylamine (XXX). Cinnamaldoxime wasreduced by the method of Ruggli & Prijs (1945) to givephenylpropylamine, b.p. 62-65°C/1.4mmHg (42.5%yield).

o-Aminomethylbenzoic acid (XLI). o-Phthalimido-methylbenzoic acid (8.4g), prepared by the method ofBornstein, Drummond & Bedell (1958), was refluxed inethanol (lOOml) and hydrazine hydrate (2ml) for 3h. Thesolid, filtered off from the reaction mixture, was extractedwith acid to give o-aminomethylbenzoic acid hydro-chloride (2.5g, 66% yield). Neutralization with ammoniagave o-aminomethylbenzoic acid.m-Aminomethylbenzoic acid (XLII). m-Cyanobenzyl-

phthalimide, prepared by the method of Reinglass (1891),was hydrolysed by refluxing with acetic acid (34%, v/v)and cone. HCI (66%, v/v) for 3 days. Phthalic acid wasextracted with ether, and m-aminomethylbenzoic acidwas isolated as the hydrochloride.p-Aminomethylbenzoic acid (XLIII). This was pre-

pared by the method of Albert & Magrath (1944).

172 1970

SUBSTRATE SPECIFICITY OF DIAMINE OXIDASE

RESULTSTable 1 lists the RF values for purified amines and

amino acids, along with an approximate evaluationof the inter-nitrogen distance for diamines. Therates of oxidation of these potential substrates arepresented as a percentage of the rate with cadaver-ine, the best-known substrate, and a comparison isgiven with values reported by previous workers,where this is available.

There is good agreement with literature values,except for compound (XI), which we found to be agood substrate, butwhich was reported by Blaschko& Chrusciel (1959) to be unoxidized, and com-pounds (XVI) and (XVII), which were not oxidizedas well as is reported (Zeller et at. 1956). Of themonoamines tested, only propylamine and butyl-amine were appreciably oxidized under the con-ditions used, there being no measurable oxygenuptake with tryptamine, tyramine, mescaline, nor-adrenaline and the phenylalkylamines (XXVIII)-(XXXI). The reported oxidation of some of thesecompounds may be due to the use of crude enzymeprepaxations.

It has been reported that C-terminal hydroxyl-ation of alkylamines improves their substratepotency (Fouts et at. 1957), but we found that thiseffect is only small. 4-Aminobutan-l-ol, 5-amino-pentan-l-ol and 6-aminohexan-1-ol were demon-strably oxidized only if the substrate concentrationwas raised tenfold. The same behaviour was shownby the series of alkylamines from hexylamine tododecylamine, which showed a slow oxygen uptakeonly in concentrated solutions, and here the in-solubility of the amines becomes a limiting factor,making accurate measurement difficult.

Table 1 also shows that none of the c-aminoacids tested was oxidized at all. Also, it is apparentthat the best substrates have inter-nitrogen dis-tances of approx. 6.0-9.0 A.

Table 3 shows the effective inhibition of theenzyme by reagents specific for carbonyl groups andthe copper-chelating agent diethyldithiocarbamate,and also the marked inhibitory power of com-pounds (IX), (X) and methylamine. The aminoacids, ammonium chloride, trimethylammoniumchloride, tetramethylammonium chloride and othermethylated amines tested were only weakinhibitors.

Table 3. Inhibition of pig kidney diamine oxidase

The substrate was 1,5-diaminopentane (3.3 mm) except in experiments marked *, wherep-bis(aminomethyl)-benzene (3.3mM) was used.

InhibitorKCNKCN*SemicarbazideSemicarbazide*HydrazineHydrazine*HydroxylamineHydroxylamine*Diethyldithiocarbamate(IX)(X)(XX)(XXI)(XXIII)(XXIV)NH4ClCH3 NH3Cl(CH3)3NHCI(CH3)4NCI(XXXVII)(XXXVIII)(XXXIX)(XL)(XLI)(XLII)(XLIII)

Concn.(mM)

1.01.00.010.010.010.010.010.011.03.33.33.33.33.33.33.33.33.33.33.33.33.33.33.33.33.3

Oxrygen uptake(,Il/min)

No inhibitor With inhibitor1.040.551.170.551.040.551.040.551.040.870.871.091.091.091.090.660.660.660.661.231.231.231.231.231.231.23

0.110

0

0

0

0

0

0

0.250.060.031.040.901.040.900.630.360.620.631.121.091.051.111.171.081.10

Relative rate(%)110000000

2473

958395839555959591898590958889

Vol. 117 173

W. G. BARDSLEY, C. M. HILL AND R. W. LOBLEY

DISCUSSION

AIny attempt to rationalize the mode of action ofthis enzyime must satisfy certain general obser-vations, listed below, which will then explain thebehaviour of any coinpouind as a substrate or in-hibitor.

Observationi 1. Only primary amines with freeoa-hydrogen atoms are oxidized. Substitutioni onthe nitrogen atoim or miethylene group causes lossof activity.

Observation 2. Diainiines are oxidized morereadily than monoamiiines, and, in the best sUb-strates, the amino groups are separated by a four- orfive-membered methylene chain, i.e. with an inter-nitrogen separation of 6.0-9.0 A.

Observation 3. The enzyme is completely in-hiibited by carbonyl-group-specific reagents andcopper-chelating agents, and is strongly inhibitedby alkylguanidines, alkyldiguanidines and someprimary amines. Secondary and tertiary aminesand co-amino acids are weak inhibitors.

Observation 4. Oxidation of alkylamnines is slowand can be demonstrated only by using concen-trated solutions ofenzyme and substrate. There is adefinite increase in rate of oxidation from inethyl-amine to butylamine and a decrease from pentyl-amnine onward. Substitution of the terminal methylgroup wvith a hydroxy substituent produces slightlybetter substrates with btutylamine, pentylainineand hexylamine.

Observation 5. Aliphatic co-amino acids and o-,m- and p-aminoethylbenzoic acids are not oxidized.

Observation 6. Diinethylaminoalkylamines andaiminoalkylguanidines, e.g. agmatine, are oxidizedat a rate abouit half that for the correspondingdiamines.

Observation 7. Arylalkylamnines, from benzyl-amine to phenylbutylamine, tyramine, noradrena-line, mescaline and tryptamnine, are very poor sub-strates. Histamnine is readily oxidized.

Observation 8. p-Bis(aminomnethyl)benzene is agood substrate, but the meta and ortho isomers,though not oxidized, are potent inhibitors.Scheme 2 is suggested as a model for substrate

binding that satisfies these observations.The binding site (BS) is best regarded as a formal

negative charge on the enzyme surface, rather thana carbonyl group as suggested by Zeller (1963),since at the optimum pH for oxidation (pH 6.4-7.8)diamnino substrates with pKa values of approx. 10.0would be present as dications and could not there-fore act as nucleophiles. The negative-chargehypothesis would then explain observation 2directly and observation 6 indirectly, since thedication from dimethylaminoalkylamines or amino-alkylguanidines could align in two possible ways,only one of them leading to oxidation. The guani-

H3N-CH2CH2CH2CH2-N

CH

(r) CU21; 02

BS Os

---6.0-9 .0 A

Scheme 2. Proposed scheme for enzyme-substrate iimter-actioii. BS is a negatively charged substrate-binding site.Hy is a substrate-binding or amino-binding site. OSrepreseiits the site for oxidation where Schiff-base for-mnation between the amino group and a pyridoxal phos-phate derivate is envisaged. deinotes strongelectrostatic attraction between BS and the second basicgroup of the substrate, which is positively charged....... illustrates the weak attraction between Hy andthe hydrophobic region of the substrate.

diino portion of an amninoalkylguanidine woutld cer-tainly be completely protonated at pH 7.0, whereasthe pyridino portion of 2-(2-aminoethyl)pyridine(pKa 3.8) would not be. This explains why, thoughagmatine is a good substrate, being bound to BS byattraction between the negative charge and thepositive charge of the substrate, 4- and 2-(2-amino-ethyl)pyridine are not oxidized, being present asmnonoamines with a nucleophilic pyridine group.Blaschko, Friedman, Hawes & Nilsson (1959) sug-gest that histamine is oxidized as the dication, sincethe pH optiimum for oxidation was 6.3. Obser-vation 5 is inost easily explained if the binding siteis negatively charged, since here the mutual repul-sion between the similarly charged enzyme andsubstrate carboxylate anion would be considerable,completelypreventingoxidation. Also, thishypothe-sis would explain why, after increasing as the chainlength is extended froin methylamine to butylamnine(observation 4) the rate falls off, since further por-tions of the alkyl chain suffer repulsion from theadjacent negative charge. This repulsion would belessened by C-terminal hydroxylation.The oxidizing site (OS) is presumed to contain

the cofactors. It seems likely that Schiff-base for-mation occurs here, followed by oxidation, and thiswould explain observations 1 and 3 (cf. Kumagai etal. 1969). Pritnary amines, e.g. methylamine and2-(2-aminoethyl)pyridine, could then be acting asinhibitors by forming Schiff bases which, for somereason, are not oxidized. Groups may be present inthe enzyme that assist in the formation of an iminebetween the pyridoxal phosphate and the primaryamino groups, e.g. a basic group could remove aproton from the -NH3+ group, or an acidic groupcould make the aldehyde more reactive. The

174 1970

Vol. 117 SUBSTRATE SPECIFICITY OF DIAMINE OXIDASE 175

combination may, however, be adequately facili-tated by the proximity into which the groups areplaced as the substrate is bound. We also suggestthat the binding and oxidizing sites are separatedby a distance of 6.0-9.0A, since this correspondsto the inter-nitrogen separation in the best diaminesubstrates (observation 2).The hydrophobic binding site (Hy) is envisaged

as binding methylene groups more strongly thanaromatic rings (observations 4 and 7). Thus theincrease in substrate potency from methylamine tobutylamine is due to progressively stronger enzyme-substrate interaction, whereas, when phenylalkyl-amine side chains are sufficiently long to interactadequately, the aromatic ring then suffers repulsionfrom thenegative charge attheamino-group-bindingsite (BS).

Several diamines (compounds VIII, IX, X andXII) with inter-nitrogen distances favourable foroxidation are not oxidized, and this information canalso be used to illuminate the nature of the hydro-phobic binding site., The case of the xylylene-diamines (observation 8) is particularly importanthere. Oxidation of the para compound (XI) showsthat there can be little steric hindrance to aromaticrings. Non-oxidation of the ortho and meta com-pounds, coupled with their considerable inhibitorypower, is difficult to explain, since here the inter-nitrogen distance is favourable for oxidation. Anexplanation is offered in Scheme 3. Here it is sug-gested that, after interacting with the amino-group-binding site strongly and the hydrophobicbinding site weakly, it is only in the para compoundthat the other amino group is juxtaposed to theoxidizing site, allowing eutopic complex-formationand oxidation to occur (cf. Zeller, 1963). With themeta and ortho isomers, dystopic complex-formation

+NH3 NH3CH2 *-- - - NH3+

Hy

CH2BS H3 * H3

Scheme 3. Plan of the interaction between bis(amino-methyl)benzenes and diamine oxidase. A eutopic enzyme-substrate complex, and thus oxidation, can only occurwith the para isomer, where the amino groups are adjacentto the binding site (BS) and oxidizing site (OS), and wherethe aromatic ring is interacting with the hydrophobicbinding site (Hy), promoting satisfactory alignment ofsubstrate and enzyme active site. Despite a favourableinter-nitrogen distance, the meta isomer can only form adystopic complex leading to inhibition but no oxidation.

would result from substrate binding, since thesecond amino group would be held away from theoxidizing site, allowing inhibition but not oxidation.A similar explanation can be given for compound(XII). Compound (VIII) probably binds to theamino-group-binding and hydrophobic bindingsites in such a way that the free amino group is heldaway from the oxidizing site, as a result ofthe bulkycyclohexane ring preventing the substrate fromlying close to the enzyme surface.

Other evidence can be presented to support thepresence of a hydrophobic binding site, e.g. the lowrate of oxidation when the methylene chain ofdiamines is hydroxylated (Zeller et at. 1956;Macholan, Rozprimovd & Sedlackova, 1967) wouldbe due to the resultant decrease in hydrophobiccharacter. The increased rate of oxidation when themethylene chain is methylated, as in 1,3-diamino-butane and 1,3-diaminopentane (Zeller, 1963),would be caused by an increase in the hydrophobiccharacter of the methylene chain of the substrate.Also, the failure of trimethylaminonium chlorideand tetramethylammonium chloride to inhibit theenzyme by ion-pair forination with the amino-binding site, suggests that, for successful in-hibition, it is necessary for binding to occur alsoat the hydrophobic binding site, as with the alkyl-guanidines and alkyldiguanidines. Finally, long-chain diamines can, of course, accommodate to therequisite inter-nitrogen distance. The poor rate ofoxidation here could be due to decreased methylenebinding in the looped conformation adopted toform a suitable enzyme-substrate complex, inaddition to the decreased population of appropri-ately spaced dications.

The authors are indebted to T. Wall and Sons (Meat andHandy Foods) Ltd. for generously supplying fresh pigkidneys. Also we thank the staff of the Department ofChemistry, University of Manchester, for determinationsof mass spectra, n.m.r. spectra and elemental analyses.

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