Isotopically sensitive regioselectivity in the oxidative deamination of a homologous series of diamines catalyzed by diamine oxidase

Chem.-Biol. Interactions, 85 (1992) 15-26

15

Elsevier Scientific Publishers Ireland Ltd.

ISOTOPICALLY SENSITIVE REGIOSELECTIVITY IN THE OXIDATIVE DEAMINATION OF A HOMOLOGOUS SERIES OF DIAMINES CATALYZED BY DIAMINE OXIDASE PATRICK S. CALLERY, BABU SUBRAMANYAM, ZHI-MIN YUAN, SOVITJ POU, LINDA A. GEELHAAR and KEVIN A. REYNOLDS

Department of Biomedicinal Chemistry, University of Maryland at Baltimore, 20 North Pine Street, Baltimore, Maryland ~1~01 (USA) (Received June 15th, 1992) (Revision received August 28th, 1992) (Accepted August 31st, 1992)

SUMMARY

The equivalence of aminomethylene groups in selected diamine substrates of diamine oxidase was exploited for the determination of intramolecular isotope effects. In the series of substrates, [1,1-2H2]-l,3-diaminopropane, [1,1-2H2]1,5-diaminopentane, [1,1-2H2]-l,6-diaminohexane, [1,1-2H2]-l,7-diaminoheptane and [~,~-2H2]-4~aminomethyl)benzylamine, the preference of the enzyme for reaction at the unlabeled methylene was found to vary from 1.45 to 10.5-fold. The observed partitioning ratios go through a minimum value with 1,5diaminopentane, the best substrate of diamine oxidase of the compounds tested. The results suggest that fast substrates have less opportunity to reorient into alternate binding conformations while bound to the active site of the enzyme. On the other hand, diamine substrates tested that cannot exist in energetically favorable conformations with internitrogen distances of about 7 - 8 ~, showed larger intramolecular isotope effects.

Key words: Intramolecular isotope effects -- Homologous substrates -- Diamine oxidase

INTRODUCTION

Porcine diamine oxidase (EC 1.4.3.6) catalyzes the oxidative deamination of a variety of diamines including 1,4-diaminobutane (putrescine) and 1,5-diaminoCorrespondence to: Patrick S. Callery, Department of Biomedicinal Chemistry, University of Maryland at Baltimore, 20 North Pine Street, Baltimore, MD 21201, USA. 0009-2797/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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pentane (cadaverine) and is a member of a family of copper amine oxidases. Isotope labeling studies on diamine oxidase [1- 6] and other copper amine oxidases, such as plasma amine oxidase [5- 8], have provided a better understanding of the stereochemistry and mechanisms involved. The binding domain at the active site of diamine oxidase appears to consist of an oxidizing site spaced from an amine-binding site by a hydrophobic binding region as first proposed by Zeller [9]. Bardsley et al. [10] estimated the optimum internitrogen distance for diamine substrates to be 6 - 9 A. This estimate was based on the measurement of interatomic distances on models of active substrates rotated to conformations in which the amino groups were separated by maximum distances. Many diamine substrates of diamine oxidase are symmetrical molecules that can be deaminated at identical terminal aminomethylene functionalities. The presence of two equivalent oxidation positions provides an opportunity to apply stable isotope methodologies to the determination of intramolecular isotope effects. For 1,4-diaminobutane labeled with deuterium on the methylene alpha to the amino group ([1,1-2H2]-l,4-diaminobutane), diamine oxidase shows a fourfold preference for the nondeuterated methylene [3,4]. In contrast, a small intramolecular isotope effect (kH/kD--1.3) was observed by Richards and Spenser [4] for the similarly labeled methylene homologue of 1,4-diaminobutane, [1,1-2H2]-l,5-diaminopentane. They suggested that the smaller intramolecular isotope effect determined for 1,5-diaminopentane was associated with limited rotational mobility once bound to the active site. Intramolecular and intrasubstituent isotope effect studies on selectively deuterated substrates have been extensively applied to cytochrome P-450 catalyzed oxidation reactions [11- 28]. Reported here are intramolecular partitioning isotope effect studies on the deamination of a series of diamine oxidase substrates varying in interatomic nitrogen distances. MATERIALS AND METHODS

Synthesis of [1,1-2H2]-l,5-diaminopentane dihydrochloride (3) Potassium phthalimide (4.0 g, 22 mmol) in 50 ml of NJV-dimethylformamide and 2.6 g (22 mmol) of 5-chloropentanonitrile were heated under reflux for 4 h. The residue, after evaporation of solvents, was dissolved in 50 ml of CHC13 and the organic layer was washed three times with water (90 ml total). The organic layer was dried (Na2SO4) and evaporated to obtain 3.4 g of crude 5phthalimidopentanonitrile (68% yield), m.p. 7 3 - 75°C. Without further purification, 5-phthalimidopentanonitrile (2.0 g, 8.7 mmol) was reduced with 2H2 (3 atm) over Pt (100 mg) in a mixture of 10 ml 2H20, 20 ml [2H]-ethanol and 3 ml of 20% 2HCI. After shaking the mixture at room temperature for 36 h, the catalyst was removed by filtration and the filtrate was diluted with 35 ml of 2 N HC1. The contents of the flask were heated under reflux for 2 h and then concentrated to a small volume under reduced pressure. On cooling to 0°C overnight, phthalic acid precipitated and was removed by filtration. The filtrate was evaporated to dryness under reduced pressure and the residue was recrystallized

17 from ethanol:water (90:10) to obtain 1.3 g (83070yield) of 3, m.p. 259- 261°C. For GC-MS analysis, the bis NgV-dimethylformamidino derivative was formed by reacting 3 with N~-dimethylformamide dimethyl acetal at 100 °C for 5 rain [29]. Mass spectrum (M +) m/z 213 (2H1, 10.8%), 214 (2H2, 89.2070).

Synthesis of [1,1-2H2]-l,7-diaminoheptane dihydrochloride (7) Compound 7 was synthesized in two steps from 7-bromoheptanonitrile as described for 3. The intermediate, 7-phthalimidoheptanonitrile, was prepared by reaction of 4.2 g (22 retool) of 7-bromoheptanonitrile with 4.0 g (22 retool) of potassium phthalimide. Yield: 4.8 g (85%), m.p.: 57-59°C. Reduction and hydrolysis of 7-phthalimidoheptanonitrile (1.0 g, 4 retool) yielded 0.45 g (56070) of 7, mp 242-243°C. Mass spectrum (M ÷) m/z 241 (2H1, 13.2%), 242 (2H2, 86.8070) as its bis Nfl/-dimethylformamidino derivative.

Synthesis of [a,a-2H2]-4-(aminomethyl)benzylamine dihydrochloride (9) 4-(Phthalimidomethyl)benzonitrile (1.0 g, 4 mmol) was reduced with 2H2 following the procedure described for the synthesis of 3 to yield 0.39 g (48%) of 9, m.p. 236-241°C. Mass spectrum (M ÷) of its bis N~N-dimethylformamidino derivative (M ÷) m/z 247 (2H1, 6.5%), 248 (2H2, 93.5%).

Synthesis of [1,1,5,5-2HJ-1, 5-diaminopentane dihydrochloride (11) Glutaronitrile was catalytically reduced with 2H2 over platinum in a mixture of 10 ml 2H20 and 4 ml of 2HC1 to give pure tetradeuterated 11, m.p. 211-212°C. The chemical ionization (methane) mass spectrum of its bis trifluoroacetyl derivative formed by the dissolution of 11 in trifluoroacetic anhydride gave (MH +) m/z 298 (2H3, 4.3070) and 299 (2H4, 95.7°70).

Synthesis of [1,1-~H2-1, 6-diaminohexane dihydrochloride (5) Reduction of 2.0 g (18 mmol) of 6-aminohexanonitrile with 2H2 gas under the conditions described for 3 excluding the hydrolysis step yielded 2.6 g (76%) of 5, m.p. 251-252°C. Mass spectrum (M+) m/z 227 (2H1, 9.807o), 228 (2H2, 90.2070) as its bis N,hr-dimethylformamidino derivative.

Synthesis of [1,1-2H2]-l,3-diaminopropane dihydrochloride (1) 3-Aminopropionitrile fumarate (1.6 g, 12.5 mmol) was reduced with 2H2 following the procedure described for compound 3 and recrystallized from ethanol (90070 crude yield). Product 1 was purified further to remove trace amounts of 3-aminopropanol hydrochloride side product by washing with cold ethanol; m.p. 246 -250°C. Mass spectrum (methane chemical ionization) of its bis trifluoroacetyl derivative (MH +) m/z 258 (2H1, 8.8%), 259 (2H2, 91.207o).

Isotopic purity of substrates Isotopic purity of the synthesized diamines was calculated as percent deuterium content from integrated mass chromatograms of at least three analyses of the individual compounds (as either a bis N~N-dimethylformamidino derivative or a bis trifluoroacetyl derivative) under identical GC-MS operating conditions. Corrections were made for 13C natural isotopic abundances measured from mass spectra of unlabeled substrates.

18

D

O

D

O

H . N ~

H + H2N/~/~D

2-d2

D

2-dr

D

H T N ~ N H

~

3

4-d2

4-dl

D D D

D 6-d 2

6-d 1

DD

H2N~',/'~',-'~',-~NH ~ ~

D D

D 7

8-d2

D

°

D

H2N

NH2 9

H~N

8-dl

+

H 10 -d2

10-dl

Scheme 1. Deuterated diamine substrates of diamine oxidase and their corresponding deuterated products.

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0

0

cF NH ofl c 3

© I COC6F 5

12

13

I

I COC6F 5

COC6F s

14

15

CF~

16 Scheme 2. Structures of derivatized metabolites of substrates of diamine oxidase that were analyzed by GC-MS.

Incubation of deuterated diamine substrates with diamine oxidase Porcine kidney diamine oxidase was used either as purchased from Sigma Chemical Co. (0.06 units/mg) or was partially purified following the method of Tamura et al. [30]. Apparent Km values for putrescine and cadaverine were of the same magnitude as literature values [31,32]. Under the incubation conditions used, further oxidation of products was not observed [29]. Incubation mixtures consisted of 10 mg (0.6 units) of diamine oxidase in 3 ml of sodium phosphate buffer (0.15 M, pH 7.4) and the dideuterated diamine substrates 3, 5 and 7 (1.5- 5.8 mM) shaken open to air at 37°C for 30-120 min. The incubations were terminated by addition of a large excess of NaBH4 (about 10 mg) and the resulting mixtures extracted with an equal volume of toluene containing 10% (v/v) pentafluorobenzoyl chloride. Small aliquots of the toluene layer were monitored by GC-MS for the N-pentafluorobenzoyl derivatives 13, 14, or 15 of the corresponding deuterated cyclic amines [33]. Ratios of the abundances of the molecular ions of the dideuterated product to monodeuterated product in the incubation mixture for each compound were calculated from integrated mass chromatograms. Control incubations were run concurrently with each tube under identical conditions in the absence of enzyme. [1,1-2H2]-l,3-Diaminopropane (5.8 mM) was incubated in water (3 ml, adjusted to pH 7.5) for 60 min with diamine oxidase (approximately 6 units) which

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had been partially purified by hydroxyapatite chromatography as described by Tamura et al. [30]. Sodium cyanoborohydride (10 mg) was present in these incubations to convert the enzymatically formed aldehyde product to 3-aminopropanol. The reaction was terminated by acidification to pH 2 with HC1 and lyophilization. The residue was extracted with acetonitrile (100 ~l) and the separated supernatant treated with trifluoroacetic anhydride (50 ~l) for 30 min at 60°C. The resulting N,O-ditrifluoroacetyl-3-aminopropanol (12) was analyzed by GC-MS for deuterium content. Control samples consisting of heat-denatured enzyme indicated the absence of artifactual 3-aminopropanol. Mono- and dideutero 4-(aminomethyl)benzaldehyde (10) formed from incubation of [~,~-2H2]-4-(aminomethyl)benzylamine with diamine oxidase were extracted from alkalanized incubation mixtures with chloroform and derivatized by addition of trifluoroacetic anhydride to the chloroform layer. The deuterium content in the resulting 4-(trifluoroacetylaminomethyl)benzaldehydes (16) was monitored by GC-MS. [1,1,5,5-2H4]-l,5-Diaminopentane (11, 2.9 mM) and unlabeled 1,5-diaminopentane (2.9 mM) were incubated together with diamine oxidase under the same conditions as described for incubations with [1,1-2H2]-l,5-diaminopentane. At the end of the 120 min incubation time, the isotopic enrichment in the product, 1-piperideine, was determined following conversion to N-pentafluorobenzoylpiperidine as described above.

Calculation of isotope effects Calculations of intramolecular isotope effects were based on the relative intensities of mass spectral fragment ions corresponding to derivatized di- and monodeuterated products. The calculated isotope effect, k H / k D w a s determined by using equation 1, which was derived using the following three premises concerning the contributions that mono- and dideuterated substrates made to the relative intensity of each m/z measurement. (i) The ratios of dideuterated and monodeuterated products obtained from the dideuterated substrates are dictated by the isotope effect, kH/kD. (ii) The ratio of monodeuterated and nondeuterated products obtained from the isotopomer of the monodeuterated substrate with deuterium located in the stereospecifically reactive position will be determined by the same isotope effect, kH/kD. (iii) The other isotopomer will give rise only to monodeuterated product. Two further assumptions were made in the derivation of equation 1. The contribution of natural abundance 13C in the monodeuterated product to the measurement of the dideuterated product is insignificant and secondary isotope effects are negligible in comparison to primary isotope effects. Equation 1:

kH

O2+

kD

/ D2 R

-

D1)

21

where D1 is the fraction of monodeuterated diamine substrate, D2 the fraction of dideuterated diamine substrate and R is the mass spectrometrically determined (M + 2)/(M + 1) ratio in the product. Intermolecular isotope effects were calculated as the ratio of the amount of product formed from [1,1,5,52H4]-1,5-diaminopentane and from unlabeled 1,5diaminopentane when incubated together in equimolar concentrations.

Molecular modeling Calculations were performed with Sybyl 5.41 software (Tripos Associates, St. Louis, MO) on the bis ammonium forms of the diamines. Initially, molecular mechanic calculations using the Tripos force field parameters were employed to locate energy minimum conformers. These structures were then subjected to full energy minimization of all geometric variables with the semi-empirical AM1 method [34] in the program MOPAC as implemented in Sybyl. Internitrogen distances were measured on the lowest energy conformer of each alkyl diamine. RESULTS

Synthesis of deuterium labeled substrates The selectively dideuterated diamine analogues, [1,1-2H2]-l,3-diaminopropane, [1,1-2H2]-l,5-diaminopentane, [1,1-2H2]-l,6-diaminohexane, [1,1-2H2]-1,7diaminoheptane and [a,a-2H2]-4-(aminomethyl)benzylamine, were synthesized either by direct catalytic reduction with deuterium gas of the corresponding aminoalkylnitrile or reduction of the phthalimidoalkylnitrile intermediates followed by acid hydrolysis. [1,1,5,5-2H4]l,5-Diaminopentane was prepared from glutaronitrile by catalytic reduction with deuterium gas.

Identification of deaminated products The 5-,6- and 7-carbon chain length diamines are oxidatively deaminated by diamine oxidase to form the corresponding 6-, 7- and 8-membered ring cyclic imine metabolites. Direct analysis of these cyclic imines for deuterium content is hampered by the tendency of these compounds to polymerize or thermally degrade. For the analysis of cyclic imine metabolites, the product of enzymatic oxidation from each incubation was chemically reduced with sodium borohydride to form the more stable cyclic amine. The resulting cyclic amines were converted to pentafluorobenzamide derivatives by an extractive acylation procedure for analysis by GC-MS [33]. Measurement of deuterium content in 3-aminopropanal formed in the incubation of deuterated 1,3-diaminopropane with diamine oxidase required a different approach from the longer chain diamines since this aminoaldehyde does not cyclize to a cyclic imine. Sodium cyanoborohydride effectively reduced enzymatically formed 3-aminopropanal to a more stable product, 3-aminopropanol. Derivatization of 3-aminopropanol with trifluoroacetic anhydride yielded a product which was efficiently analyzed by chemical ionization GC-MS. Deuterated 4-aminomethylbenzaldehyde formed in the deamination of labeled 4-(aminomethyl)benzylaminewas extracted with chloroform and detected by GC-

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MS after derivatization with trifluoroacetic anhydride. Deuterium content was determined on the resulting 4-(trifluoroacetylaminomethyl)benzaldehyde (16).

Int~'a~nolecula~" isotope effects All incubations of diamines with diamine oxidase were at saturating concentrations of approximately 10-fold greater than the Km values [31,32] which assumes that all of the enzyme present is in an enzyme-substrate complex and that initial binding is not being considered. Calculated intramolecular isotope effects for six substrates of diamine oxidase are given in Table I. The largest isotope effect was observed for 1,3-diaminopropane, which is the poorest substrate of diamine oxidase in the series tested as seen from data generated by Bardsley et al. [10] on the relative oxidation rates of diamines (Table I). In contrast, the smallest isotope effect was found for the best substrate, 1,5diaminopentane. Variations in experimental conditions made by changing the pH of the incubation mixture to pH 5 or 8, shortening the incubation time to 30 min and reducing the concentration of substrate to 1.5 mM had little influence on the observed isotope effect.

TABLE I INTRAMOLECULAR ISOTOPE E F F E C T S IN THE DEAMINATION OF DEUTERATED DIAMINE SUBSTRATES WITH DIAMINE OXIDASE Substrate

(M + 1) ÷/(M + 2) + m/z ratio observeda

kH]k D (2H2/2H1) calculated b

[1,1-2H2]-1,3 Diaminopropane [1,1-~H2]-1,4 Diaminobutane |1,1-2H2]-1,5 Diaminopentane [1,1-2Hz]-l,6 Diaminohexane [1,1-ZH2]-l,7Diaminoheptane [a,,~-2H~]-4-(Amino methyl)benzylamine

5.1 ± 0.2

10.50 ± 2.3 (3)

Internitrogen distance c (~)

Relative oxidation rate d

5.04

8

--

4.44 ± 0.05 (4) c

6.31

96

1.29 ± 0.06

1.45 ± 0.07 (3)

7.53

100

1.56 ± 0.08

1.62 :e 0.09 (6)

8.79

41

1.52 ± 0.11

1.93 ± 0.17 (4)

10.02

43

1.33 ± 0.06

1.53 ± 0.08 (3)

7.45

48

aValues represent mean ± S.D. of the number of experiments in parentheses and are the observed ratios of mono- and dideutero products. bCorrected for contributions from incompletely deuterated substrates using equation 1 in text. eInternitrogen distances were calculated on energy-minimized conformers of fully protonated diamines. dData are percentages of 1,5-diaminopentane activity from Bardsley et al. [10]. CData from Callery et al. [3].

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Inte,'molecular isotope effects To determine the intermolecular isotope effect of deuterium substitution of all four a-hydrogens, tetradeuterated 1,5-diaminopentane was prepared. This symmetrically deuterated substrate cannot reveal intramolecular isotope effects. A comparison of the rate of formation of product arising from deamination of [1,1,5,5-2H4]-l,5-diaminopentane with the rate of product formed from unlabeled 1,5-diaminopentane incubated together in equimolar concentrations gave an estimated kH/kD of 1.82 ± 0.11 expressed as the mean of five determinations. Molecular modeling Since the diamines are expected to be protonated at physiological pH, all calculations were performed on the bis ammonium forms using the AM1 semiempirical method [34]. Although there were many conformers calculated with nearly equal energy, the lowest energy conformers of the protonated diamines were in the fully-extended chain form. In the case of the rigid analogue, 4(aminomethyl)benzylamine, two conformers of low energy were found, a cis conformer with the ammonium groups on the same side of a face of the phenyl and a trans conformer of slightly lower energy. In Table I are listed the calculated internitrogen distances for the protonated diamines tested. DISCUSSION When a diamine substrate deuterated on the carbons of one aminomethylene is incubated with diamine oxidase, products can result from the oxidation of either terminal methylene. If the distribution of the two products (monodeuterated and dideuterated) were governed entirely by probability, both products would form in equal amounts. Deviation in this distribution from unity is indicative of the preference of the enzyme for oxidation of one end of the molecule over the other resulting in an intramolecular isotope effect. In the series of deuterated substrates tested here, the magnitude of this preference exhibited by diamine oxidase in the oxidation of diamine substrates was determined on compounds unsymmetrically deuterated at one end of the molecule in which the hydrogens of one of the methylene groups alpha to the amine moieties were replaced with deuterium. The intramolecular deuterium isotope effect (kH/kD ratio) for the deamination of diamines of varying chain length by diamine oxidase was calculated as a ratio of dideuterated to monodeuterated product (2H2/2H1). As indicated in Table I, there is a striking difference in the magnitude of intramolecular deuterium isotope effects between [1,1-2H2]-l,3-diaminopropane and the higher homologues studied. In these experiments, the deuterium labeled terminus of the diamine molecules was found to be oxidized at a slower rate than the nondeuterated methylene as indicated by a positive kH/kD ratio for the entire series studied. The choice of dideuterated isotopomers as substrates did not allow for an assessment of the stereochemistry of the oxidation or for secondary isotope effects resulting from the presence of a deuterium at a bond not broken in the reaction [4,5]. Intramolecular isotope effects for the deamination reaction were less pro-

24

nounced in chains longer than four methylenes, ranging from 1.45 to 1.93. Data from these experiments indicate that there is a notable shift in the relative importance of unknown contributing steps to the overall rate-limiting step in moving from 1,3-diaminopropane to 1,5-diaminopentane and higher homologues. Although the chemical mechanism and the transition state structures cannot be known without determination of the intrinsic isotope effect and other mechanistic studies, these results agree with the interpretation made by Richards and Spenser [4] regarding the inconsistency of a single unifying mechanism for the oxidative deamination of aliphatic diamines by diamine oxidase. For [1,1-2H2]-l,5-diaminopentane, the intramolecular isotope effect on the deamination reaction is much smaller ( k H ] k D = 1.45) than the shorter-chain substrates indicating that the binding equilibrium step is slower than that for [1,1-2H2]-l,3-diaminopropane or [1,1-2H2]-l,4-diaminobutane. The intermolecular isotope effect ( k H / k D --- 1.82) obtained for the tetradeuterated substrate, [1,1,5,5-2H4]-l,5-diaminopentane, was also a small number in a similar fashion to that observed for tetradeuterated 1,4-diaminobutane [3]. Intermolecular isotope effects for the other homologues studied were not determined. The intramolecular isotope effect for the deamination of 1,6-diaminohexane and 1,7-diaminoheptane are larger than that for 1,5-diaminopentane indicating that there is less suppression of the isotope effect for these deamination reactions in comparison to that of 1,5-diaminopentane resulting perhaps from decreased binding affinity with increased chain length. This suggests that substrates of length or size differing from that of a near ideal binding site distance exemplified by the internitrogen distance in 1,5-diaminopentane dissociate from the catalytic surface more readily. Data in Table I indicate that the observed intramolecular isotope effects for 1,6-diaminohexane and 1,7-diaminoheptane are slightly larger than that for 1,5diaminopentane and that there is not a linear relationship between chain length and intramolecular isotope effect in the series tested. This perturbation from linearity can be explained on the basis of possible decreased interaction between the longer-chain substrate molecules with the enzyme associated with folding of the polymethylene regions of these oversized substrates into a conformer which has the appropriate internitrogen distance. A rigid analogue of the polymethylene diamines, [~,a-2H2]-4-(aminomethyl) benzylamine, was evaluated. In this substrate of diamine oxidase [10], the two amino groups are held apart by a phenyl spacer and the terminal amines are conformationally restricted in an equivalent of the fully extended conformer of the alkyl chain diamines. Modeling of this structure indicated that a low energy conformation exists in which the amines are 7.45 A apart. This distance most closely resembles the distance calculated for 1,5-diaminopentane (7.53 A), which is the substrate size that showed the lowest intramolecular isotope effect of the diamines tested. In keeping with an apparent internitrogen distance relationship with isotope effect magnitude, [~,a-2H2]-4(aminomethyl)benzylamine yielded a small intramolecular isotope effect (kI-i/kD = 1.53). Although internitrogen distance correlated with intramolecular isotope effect magnitude, the oxidation rates as reported by Bardsley et al. [10] did not correlate well with isotope effect

25 for 4-(aminomethyl)benzylamine. Whereas 4-(aminomethy)benzylamine shares a s i m i l a r i n t e r n i t r o g e n d i s t a n c e w i t h 1 - 5 - d i a m i n o p e n t a n e , it is o x i d i z e d a t a r a t e l e s s t h a n h a l f t h a t o f 1 , 5 - d i a m i n o p e n t a n e [10]. A n a l t e r n a t e e x p l a n a t i o n f o r t h e o b s e r v e d s m a l l i s o t o p e e f f e c t o b t a i n e d f o r 4( a m i n o m e t h y l ) b e n z y l a m i n e could b e t h a t t h e s t e r i c b u l k a s s o c i a t e d w i t h i t s p h e n y l m o i e t y h i n d e r s r e o r i e n t a t i o n o f t h i s s u b s t r a t e o n c e b o u n d to t h e e n z y m e . T h e r e s u l t s s u g g e s t t h a t a n i n t e r n i t r o g e n d i s t a n c e a p p r o a c h i n g t h a t o f 1,5d i a m i n o p e n t a n e r e p r e s e n t s t h e o p t i m u m d i s t a n c e b e t w e e n t h e o x i d i z i n g site a n d t h e a m i n e - b i n d i n g s i t e on t h e s u r f a c e o f d i a m i n e o x i d a s e . T h i s c o n c l u s i o n is b a s e d on t h e h i g h l y s u p p r e s s e d i n t r a m o l e c u l a r i s o t o p e e f f e c t s f o r 1 , 5 - d i a m i n o p e n t a n e a n d i t s r i g i d a n a l o g u e in c o m p a r i s o n to o t h e r d i a m i n e s u b s t r a t e s . REFERENCES 1 W.G. Bardsley, M.J.C. Crabbe and J.S. Shindler, Kinetics of the diamine oxidase reaction, Biochem. J., 131 (1973) 459-469. 2 J.C. Richards and I.D. Spenser, 2H NMR as a probe of the stereochemistry of enzyme reactions at prochiral centers. Deamination of cadaverine catalyzed by diamine oxidase, J. Am. Chem. Soc., 100 (1978) 7402-7404. 3 P.S. Callery, M.S.B. Nayar, E.M. Jakubowski and M. Stogniew, Intermolecular and intramolecular isotope effects in the deamination of putrescine catalyzed by diamine oxidase, Experientia, 38 (1982) 431-433. 4 J.C. Richards and I.D. Spenser, 2H NMR spectroscopy as a probe of the stereochemistry of enzymic reactions at prochiral centres, Tetrahedron, 39 (1983) 3549-3568. 5 P.H. Yu, Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases, Biochem. Cell Biol., 66 (1988) 853-861. 6 A.A Coleman, O. Hindsgaul and M.M. Palcic, Stereochemistry of copper amine oxidase reactions, J. Biol. Chem., 264 (1989) 19500-19505. 7 M. Farnum, M. Palcic and J.P. Klinman, pH dependence of deuterium isotope effects and tritium exchange in the bovine plasma amine oxidase reaction. A role for single-base catalysis in amine oxidation and imine exchange, Biochem., 25 (1986) 1898-1904. 8 C. Hartmann and J.P. Klinman, Structure-function studies of substrate oxidation by bovine serum amine oxidase: relationship to cofactor structure and mechanism, Biochem., 30 (1991) 4605-4611. 9 E.A. Zeller, R. Stern and M. Wenk, Uber die diamin-diamin-oxydase-reaktion,Helv. Clin. Acta, 23 (1940) 3-17. 10 W.G. Bardsley, C.M. Hill and R.W. Lobley, A reinvestigation of the substrate specificity of pig kidney diamine oxidase, Biochem. J., 117 (1970) 169-176. 11 A.B. Foster, M. Jarman, J.D. Stevens, P. Thomas and J.W. Westwood, Isotope effects in O: and N-demetylations mediated by rat liver microsomes: an application of direct insertion electron impact mass spectrometry, Chem.-Biol. Interact., 9 (1974) 327-340. 12 M.M. Abdel-Monem, Isotope effects in enzymatic Nodemethylation of tertiary amines, J. Med. Chem., 18 (1975) 427-430. 13 L.M. Hjelmeland, L. Aranow and J.R. Trudell, Intramolecular determination of primary kinetic isotope effects in hydroxylations catalyzed by cytochrome P-450, Biochem. Biophys. Res. Commun., 76 (1977) 541-549. 14 J.T. Groves, G.A McClusky, R.E. White and M.J. Coon, Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. Evidence for a carbon radical intermediate, Biochem. Biophys. Res. Commun., 81 (1978) 154-160. 15 G.T. Miwa, W.A Garland, B.J. Hodshon, A.Y.H. Lu and D.B. Northrup, Kinetic isotope effects in cytochrome P-450 catalyzed oxidation reactions, J. Biol. Chem., 255 (1980) 6049-6054.

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