Effect of coupling site and nature of the polymer on the coenzymatic properties of water-soluble macromolecular NAD derivatives with selected dehydrogenase enzymes

Sergio Riva and Giacomo Carrea*

lstituto di Chimica degli OrmonL CN.R., Via Mario Bianco 9, 20131 Milan, ltaly

and Francesco M. Veronese

Dipartimento di Scienze Farmaceutiche (Centro di Chimica del Farmaco e deiProdotti Biologicamente A ttivi, C.N.R.), 35100 Padua, Italy

and Andreas F. Biickmann

Gesellschaft far Biotechnologische Forsehung, Mascheroder Weg 1, D-3300, Braunschweig, FRG

(Received 4 March 1986)

The effects of the position of coupling to NAD ÷ (N-l, N 6 or C-8 of the adenine ring) and the nature of the polymer [(poly(ethylene glycol), poly(ethylenimine)and poly(acrylicacid)] on the coenzymatic properties of water-soluble macromolecular NAD ÷ derivatives have been investigated systematically. The enzymes selected for the study were two hydroxysteroid dehydrogenases, used for the synthesis of bile acids of pharmaceutical interest, and glutamate dehydrogenase, formate dehydrogenase and glucose dehydrogenase, which most effectively regenerate NAD(P)÷,'NADH or NAD(P)H, respectively. It was found that the N 6 position of the adenine ring and neutral poly(ethylene glycol) were the site and polymer giving the most satisfactory results for the majority of these enzymes. Glutamate dehydrogenase, formate dehydrogenase and 3~-hydroxysteroid dehydrogenase with poly(ethylene glycol)-N6-(2-aminoethylJ-NAD ÷ had V values which were 39, 57 and 66% of those with NAD ÷

m a x

and K m values 3.5, 5.5 and 1 7 times those with NAD ÷. No derivatives had good activity with glucose dehydrogenase and 7~-hydroxysteroid dehydrogenase.

Keywords: Kinetics; macromolecular NAD+; oxidoreductases; poly(ethylene glycol); poly(ethylenirnine); poly(acrylic acid)

Introduction

The possibility of using NAD-dependent dehydrogenases for synthesis or modification of compounds of practical interest is of particular importance because of the great stereo- and regiospecificity of these enzymes. 1-7 The trans- formations are usually carried out in batch reactors but, for industrial-scale syntheses, the enzymatic system based on continuous-flow membrane reactors with simultaneous enzymatic regeneration of NAD(H) seems to be the most promising. 2's-ll In such a system the coenzymes must be in a macromolecular form in order to be retained inside the reactor.

A great variety of water-soluble macromolecular NAD ÷ derivatives have been prepared and their coenzymatic pro- perties tested with enzymes of little practical interest, 12-~9

*To whom correspondence should be addressed

except for a few cases regarding the synthesis of amino acids. 8'9'2° In the present work we systematically investi- gated the effects of the position of coupling to NAD ÷ (N-l, N 6 or C-8 of the adenine ring) and the nature of the soluble polymer (neutral, acidic or basic) on the coenzymatic properties of the NAD ÷ analogues. The enzymes selected for the study were two specific hydroxysteroid dehydro- genases used for the synthesis of bile acids of pharmaceutical interest 4,2t and glutamate dehydrogenase, formate dehydro- genase and glucose dehydrogenase, which most effectively regenerate NAD(P) 7,22, NADH 2,4'2a or NAD(P)H 1'24 respectively.

Materials and methods

Materials

NAD ÷ i(grade I), yeast alcohol dehydrogenase (alcohol: NAD ÷ oxidoreductase, EC 1.1.1.1), formate dehydro- genase (formate:NAD ÷ oxidoreductase, EC 1.2.1.2) and

0141 --0339/86/090556--05 $03.00 556~ Enzyme Microb. Technol., 1986, vo] 9, September © 1986 Butterworth & Co. (Publishers) Ltd

glutamate dehydrogenase (L-glutamate:NAD(P) ÷ oxidore- ductase, EC 1.4.1.3) were purchased from Boehringer. 3o~-Hydroxysteroid dehydrogenase (3a-hydroxysteroid: NAD(P) ÷ oxidoreductase, EC 1.1.1.50), 7a-hydroxysteroid dehydrogenase (7a-hydroxysteroid:NAD ÷ oxidoreductase, EC 1.1.1.159) and glucose dehydrogenase O-o-glucose: NAD(P) ÷ oxidoreductase, EC 1.1.1.47) were obtained from Sigma. Poly(ethylene glycol) (PEG, tool. wt 20000) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (CDI) came from Fluka and monomethoxypoly(ethylene glycol) (MPEG, mol. wt 5000), poly(ethylenimine) (PEI, mol. wt 50000-60000) , poly(methylacrylate)(tool, wt 31000) and 2,4,5-trichlorophenyl chloroformate from Aldrich. lodoacetic acid was purchased from Merck and ethylenei- mine from Serva. Bio-Rex 70 (200-400 mesh) was a Biorad product and DEAE-cellulose DE32 (microgranular DEAE) was obtained from Whatman. All other reagents and com- pounds were of analytical grade.

A naly tical procedures

Thin-layer chromatography was carried out on silica gel 60 F2s4 (Merck) in isobutyric acid/H2 0/30% aqueous NH3 (66/33/1). The dinucleotide spots were detected under an ultraviolet lamp (254 nm) and by spraying with ninhydrin, when applicable. Ultraviolet spectra were obtained with a Cary 16 recording spectrophotometer. Bromination of NAD ÷ was monitored by 1H n.m.r, with aPerkin-Elmer R12 spectrometer.

P o l y m e r preparation

PEG was carboxylated at the terminal ends (85% yield) as described by Btickmann et al. 2s MPEG was activated at the terminal end with 2,4,5-trichlorophenyl chloroformate (80% yield) as described by Veronese et al. 26 Poly(acrylic acid) (PAA) was prepared by refluxing poly(methylacrylate) with 10% NaOH for 8 h followed by neutralization with 4 M HC1, dialysis and lyophilization. Hydrolysis was about 100%, as determined by titration using phenolphthalein as an indicator.

Synthes i s o f PEG-N(1) - (2 -aminoe thy l ) -NAD ÷, PEG- N 6 - (2 -aminoe thy l ) -NAD ÷ and MPEG-N 6-(2-amino- e t h y l ) - N A D ÷

NAD ÷ (1.5 g) was reacted with ethyleneimine and purified on Bio-Rex 70, as described by B~ickmann et al. 2° The yield of pure N(1)-(2-aminoethyl)-NAD ÷ was 0.5 g (31%). PEG-N(1)-(2-aminoethyl)-NAD ÷ was prepared by condensing 3 g (0.15 retool) of carboxylated PEG with 210 mg (0.3 mmol) of N(1)-(2-aminoethyl)-NAD ÷ in the presence of CDI at pH 4.8 and purified by gel filtration on Sephadex G-50. 2° The product (2.8 g) contained about 1.35 tool NAD÷/mol polymer.

The N(1)-derivative (1.85 g) was reduced with Na2 $2 04 and rearranged to give PEG-N~/-(2-aminoethyl)-NADH, which had an A267/A33s value of 3.3, very close to that (3.2) in the literature. 2° The NADH derivative was then enzymati- cally oxidized to PEG-N ~-(2-aminoethyl)-NAD÷. 2° The der- ivative (1.8 g) contained about 1.3 tool NAD*/mol polymer.

For the preparation of MPEG-N6-(2-aminoethyl)-NAD ÷, 600 mg (0.12 mmol) of MPEG activated with 2,4,5-tri- chlorophenyl chloroformate were reacted with 70 mg (0.1 mmol) of N(1)-2(aminoethyl)-NAD ÷ in 14 ml of 0.2 M borate buffer, pH 8.8, until the content of amino groups, determined with the trinitrobenzenesulphonate method, 27 was constant (approx. 6 h). Glycine (30 mg) was added to

Coenzyrnatic properties of rnacrornolecular NAD*: S. Riva et al.

block the unreacted groups on activated MPEG and after 2 h the reaction mixture was gel filtered through a Sepha- dex G-50 column equilibrated with H20. The MPEG-N(1)- 2(aminoethyl)-NAD ÷ obtained was converted to the N 6 derivative as described above, gel filtered and lyophilized. The derivative (530 mg) contained about 0.55 mol NAD+/ mol polymer.

Syn thesis o f PEG-C(8)- (6-aminohexyl ) -amino- N A D ÷

C(8)-(6-Aminohexyl)-amino-NADH was synthesized by the method of Lee et al. 28 starting from 1 g NAD ÷, and purified by ion exchange chromatography on a DE32 column (carbonate form, 2.5 x 40 cm) eluted with a linear ammonium carbonate gradient (0-0.8 M, 1.6 litres). Before chromatography, the reaction mixture was poured into 200 ml of cold ethanol and the precipitated product collected by centrifugation and redissolved in water. This step, not described in the original procedure, 28 was necessary to obtain good chromatographic resolution. After prolonged lyophilization to remove ammonium carbonate, the NADH derivative was enzymatically oxidized to C(8)-(6-amino- hexyl)-amino-NAD ÷ as previously reported 28 and the yield of pure product was 105 mg (9%). Carboxylated PEG (1.1 g, 0.06 retool) and C(8)-(6-aminohexyl)-amino-NAD ÷ (85 mg, 0.11 mmol) were dissolved in 5 ml water. The pH was adjusted to 7 with NaOH and then 40 mg of CDI were added. The pH was maintained at 7 and after 6 h of reaction additional CDI (40 mg) was added. After 24 h of reaction at room temperature, the mixture was gel filtered through a Sephadex G-50 column equilibrated with water to separ- ate PEG-C(8)-(6-aminohexyl)-amino-NAD ÷ from low mole- cular weight compounds. The macromolecular NAD ÷, re- covered by lyophilization (1 g), contained about 0.9 tool NAD*/mol polymer.

Synthes is o f PEI-N 6 - c a r b o x y m e t h y l - N A D ÷

NS-Carboxymethyl-NAD ÷ was synthesized as described by Lindberg et al., 13 starting from 800 mg NAD ÷, and purified on a DE32 column (carbonate form, 2.5 x 40 cm) eluted with a linear ammonium bicarbonate gradient (0-0.8 M, 2 litres). After prolonged lyophilization, 110 mg (13% yield) of pure NAD ÷ derivative were obtained. N 6-' Carboxymethyl-NAD ÷ (95 mg, 0.13 mmol)'was condensed with PEI (300 mg, 5.5 /lmol) as described by Zappelli et al. as and purified, as PEIdV 6-carboxymethyl-NAD ÷ (I 55 mg) contained about 8 mol NAD+/mol polymer.

Synthes is o f P A A - N 6 - (2-aminoethy l ) -NAD*

PAA (400 rag, 13/amol) and N(1)-(2-aminoethyl)-NAD* (80 rag, 0.11 mmol) were dissolved in 5 ml water, the pH was adjusted to 5 and then 95 mg CDI, dissolved in 0.8 ml water, were added slowly. After 5 h of reaction, the solu- tion was gel filtered through a Sephadex G-50 column equilibrated with water. Macromolecular NAD ÷ was reduced with Na2 $2 04 and rearranged to PAA-N 6-(2-aminoethyl)- NADH, which had an A26,7/A335 value of 4, larger than the theoretical one (3.2). This suggests that part of the cofactor was not reduced and therefore it was degraded by the basic medium in which the rearrangement was carried out. The compound was then enzymatically oxidized, gel filtered through a Sephadex G-50 column and lyophilized. PAA- N ~-(2-aminoethyl)-NAD ÷ (250 mg) contained about 1.8 tool NAD*/mol polymer.

Enzyme Microb. Technol., 1986, vol 9, September 557

Papers H NH2

~NAI~., ~ R

PEG-N(1 )- (2 - aminoethyl)- NAD +

N

R

PEG- 6"(8)-( 6 - aminohexyl } amino - NAD +

I,~.~ N,~N/fIN I R

PEG( or MPEO) -A/6- ( 2 -aminoethyl ) - NAD +

Figure 1

R

PE I - N 6_ car boxymethyl - NAD +

Soluble macromolecular derivatives of NAD +

H N ~ H \ ( ~

I R

P A A - N 6- (2-aminoethyl) - NAD +

Enzyme assays Assays were carried out at 25°C in 1 ml cuvettes, 1 cm

path length, with spectrophotometric monitoring at 340 nm. The conditions for the various assays were as follows: formate dehydrogenase in 0.1 M potassium phosphate buffer, pH 7.5, containing 0.1 M formate; glutamate dehydrogenase in 0.1 M potassium phosphate buffer, pH 8, containing 0.01 M glutamate and 1 mM ADP; glucose dehydrogenase in 0.1 M potassium phosphate buffer, pH 7.5, containing 0.02 M glucose; 3a-hydroxysteroid dehydrogenase and 7a-hydroxysteroid dehydrogenase in 0.1 M potassium phos- phate buffer, pH 9, containing 1 m s cholate. With PEI derivatives, 0.1 M Tris buffer was used instead of phosphate. The concentrations of nicotinamide cofactors were: NAD ÷ 10-1000 /aM; PEG-N(1)-(2-aminoethyl)-NAD ÷, PEG-N 6- (2-aminoethyl)-NAD ÷, MPEG-N6-(2-aminoethyl)-NAD ÷ and PEG-C(8)-(6-aminohexyl)-amino-NAD ÷ 30-2200 /aM; PEI- N6-carboxymethyl-NAD ÷ 10-1000/aM ; PAA-N 6-(2-amino- ethyl)-NAD ÷ 30-1000 /aM. K m and Vma x values were obtained from the initial rate measurements, using the equa- tions of Wilkinson 29 programmed on an Apple IIc com- puter, a°

Results and discussion

Synthesis o f macromolecular N A D ÷ derivatives

The macromolecular derivatives of NAD ÷ synthesized are shown in Figure 1. In the first series of compounds, the same macromolecule (PEG) was linked to different positions on the adenine ring N-l, N 6 or C-8). In the second series, different macromolecutes - the neutral PEG or MPEG, the basic PEI or the acidic PAA - were linked to

the same position on the adenine ring (N 6). The functional- ization of NAD ÷ and the preparation of the majority of the macromolecular derivatives were carried out by already published procedures. 13,1s,2o,2s

The derivatives PEG-C(8)-(6-aminohexyl)-amino-NAD ÷ and PAA-N6-(2-aminoethyl)~NAD ÷ were new compounds and MPEG-N6-(2-aminoethyl)-NAD ÷ was also prepared by a new procedure based on tile single-step activation of PEG with 2,4,5-trichlorophenyl chloroformate. It should be emphasized that CDI-promoted condensation between C(8)- (6-aminohexyl)-amino-NAD + and carboxylated PEG was optimum at pH 7, unlike N(1)-(2-aminoethyl)-NAD ÷, for which the optimum was at about pH 5. 20 MPEG-N 6- (2-aminoethyl)-NAD ÷ was obtained with similar yields but in a simpler way than PEG-N 6 -(2-aminoethyl)-NAD ÷, which was prepared as described by B~ickmann and Morr 2s and required three steps for polymer functionalization. The stability of the linkage between MPEG and NAD ÷ was high and coenzyme leakage was negligible after one week of incubation of the derivative at pH 7, 25°C.

The functionalized NAD + products used for condensation with the polymers were all chromatographically purified and the yields of coenzymatically active macromolecular- NAD ÷ were greater than 85%, except with PAA-Ar6-(2- anainoethyl)-NAD* for which it was about 80%.

Comparing the procedures employed to prepare the various macrom0t,ecular NAD ÷ derivatives, the less laborious methods and those giving the highest yields were, at least in our hands, those utilized for the synthesis of PEG-N 6- (2-aminoethyl)-NAD ÷ and MPEG.Ar6 .(2.aminoethyl).NAD ÷. The most crucial step in all cases, and particularly with C(8)-(6-aminohexyl)-amino-NAD +, was the synthesis and purification of the functionalized NAD +. In the case of

5 5 8 Enzyme Microb. Technol . , 1986 , vol 9, September

PEI-N6-carboxymethyl-NAD *, the condensation step was also unsatisfactory, mainly because of the difficulty of removing the unbound NAD* from the macromolecular coenzyme, which thus reduced the yield. Furthermore, this derivative showed a tendency to aggregate and pre- cipitate during prolonged incubation in solution.

Coenzymatic properties o f macromolecular N A D ÷ derivatives

Tables 1-5 show the Km and Vmax values for several dehydrogenases with the various NAD* derivatives. To study the effects of the coupling site on the coenzymatic properties of the macromolecular coenzymes, the neutral hydrophilic PEG was linked to three different positions on the adenine ring (N-l, N 6 or C-8).

With PEG-C(8)-(6-aminohexyl)-amino-NAD*, the Vmax values were between 20 and 69% of those with NAD*, depending on the enzyme, with the exception of formate dehydrogenase which was practically inactive. However, all the K m values were higher than millimolar and this makes the derivative not very suitable for practical use. With PEG-N(1)-(2-aminoethyl)-NAD*, the enzymes were barely active, with the exception of glutamate dehydro- genase, which displayed 15% of its activity with NAD* and a fourfold increase in the Km value. The partial activ- ity of glutamate dehydrogenase is attributable to the fact that the enzyme is also NADP* dependent and several NADP*-dependent enzymes have been found to be active with N(1)-modified NADP*. 31 With PEG-N6-(2-amino - ethyl)-NAD*, the formate dehydrogenase, glutamate

Coenzymatic properties of macromolecular NA D+: S. R iva et al.

dehydrogenase and 3a-hydroxysteroid dehydrogenase had satisfactorily high Vmax values (39-66% of those with NAD*). In addition, the Km values, even though higher (3 -17 times) than those with NAD +, were lower than millimolar and therefore within a concentration range probably usable in continuous-flow membrane reactors. The coenzymatic properties of MPEG-N6-(2-aminoethyl) - NAD* were practically the same as those of PEG-N~-(2- aminoethyl)-NAD*.

Regarding the influence of the polymer nature, the basic PEI-N ~-(2-aminoethyl)-NAD* was found to possess unsatis- factory coenzymatic properties except with formate dehy- drogenase (Table 1), for which its Vmax value was 85% of that with NAD ÷ and the K m value even lower (three times)

Table 3 Kinetic constants of glucose dehydrogenase for macro- molecular NAD ÷ derivatives

Coenzyme K m (/~M) Vmax, relative

NAD* 64 100

PEG-C(8)-(6-aminohexyl)-amino- 2410 20 NAD*

PEG-N(1 )-(2-aminoethyl)-NAD* 522 0.1

PEG-N 6 ,(2-aminoethyl)-NAD* 2030 3

PEI-N 6 -carboxymethyI-NAD + 26 6

PAA-N 6 -(2-aminoethyl )-N AD ÷ n .d. n.d.

n.d., not detectable

Table 1 Kinetic constants of formate dehydrogenase for macro- molecular NAD* derivatives

Coenzyme K m (/4M) Vmax, relative

NAD* 15 100

PEG-C-(8)-(6-aminohexyl)-amino- 2450 3 NAD*

PEG-N( 1 )-(2-aminoethyl)-NAD* 1290 1

PEG-N 6 -(2-aminoethyl)-N AD+ 82 57

MPEG-N ~ -(2-aminoethyl)-NAD* 86 56

PEI-N 6 -carboxymethyI-N AD* 5 85

PAA-N ~ -(2-aminoethyl)-NAD* 86 9

Table 4 Kinetic constants o÷ 3e-hydroxysteroid dehydrogenase for macromolecular NAD + derivatives

Coenzyme K m ( # i ) Vmax, relative

NAD + 38 100

PEG-C(8)-(6-aminohexyl)-amino- 6300 51 NAD*

PEG-N(1 )-(2-aminoethyl)-NAD + 3550 4

PEG-N ~ -(2-aminoethyl)-NAD* 647 66

MPEG-N ~ -(2-aminoethyl)-NAD + 610 64

PE I-N 6 ~carboxy methyI-N AD + 108 8

PAA-N ~ -(2-aminoethyl)-NAD+ n.d. n.d.

n.d., not detectable

Table 2 Kinetic constants of glutamate dehydrogenase for macro- molecular NAD + derivatives

Table 6 Kinetic constants of 7¢x-hydroxysteroid dehydrogenase for macromolecular NAD ÷ derivatives

Coenzyme K m (#M) Vmax, relative Coenzyme K m (/~M) Vmax, relative

NAD ÷ 203 100

PEG-C-(8)-(6-a minohexyl)-amino- 3190 69 NAD +

PEG-N(1 )-(2-amin oethyl) -NAD + 849 15

PEG-N 6 -(2-aminoethyl)-NAD* 719 39

MPEG-/V 6 -(2-aminoethyl)-NA D* 680 41

PE I-N~ -carboxymet hyI-N AD + 75 4

PAA-N 6 -(2-aminoethyl)-NAD + n.d. n.d.

n .d., not detectable

N A D* 363 100

PEG-C(8)-(6-aminohexyl)-amino- 3810 20 NAD*

PEG-N(1 )-(2-aminoethyl)-N AD* 2870 0.2

PEG-N ~ -(2-aminoethvl)-NAD+ 4630 12

MPEG-N 6 -(2-aminoethyl)-NAD* 4220 13

PEI-N 6 <arboxymethyI-NAD* 1500 9

PAA4V ~ -(2-aminoethyl)-NAD + n.d. n.d.

nod., not detectable

Enzyme M ic rob . Techno l . , 1986 , vol 9 , Sep tember 5 5 9

Papers

than that with the unmodified cofactor. The acidic PAA- N 6-(2-aminoethyl)-NAD ÷ also showed coenzymatic activity only with formate dehydrogenase (Vma x value 9% of that with NAD*). The poor results obtained with the charged polymers cannot be at tr ibuted, at least in the case of PEI- N6-(2-aminoethyl)-NAD *, to electrostatic repulsions be- tween the polymer and charged groups on the enzymes because the PEI derivative had K m values lower than those of NAD ÷ not only with formate dehydrogenase but also with glutamate dehydrogenase (Table 2) and glucose dehy- drogenase (Table 3). It should be emphasized that these polymers did not inhibit the activity of the enzymes.

With glutamate dehydrogenase, 3c~-hydroxysteroid dehy- drogenase and 7c~-hydroxysteroid dehydrogenase, a macro- molecular derivative of NADH, i.e. PEG-N 6-(2-aminoethyl)- NADH was also tested. The results were in agreement with those for PEG-N6-(2-aminoethyl)-NAD ÷, since only gluta- mate dehydrogenase and 3a-hydroxysteroid dehydrogenase showed good activity (62% and 68% of those with unmodi- fied NADH), with moderate increases in the Kma x values (4 and 13 times).

In conclusion, systematic investigation of the influence of the site of coupling and nature of the polymer on the kinetic properties of macromolecular NAD ÷ showed that the N 6 position of the adenine ring and the neutral PEG or MPEG were the site and polymers giving the most satisfactory results with the majority of the enzymes tested. Further- more, (M)PEG-N 6-(2-aminoethyl)-NAD ÷ was also the deriva- tive with the highest yield and the least laborious prepara- tion. However, no derivative had good activity with glucose dehydrogenase or 7c~-hydroxysteroid dehydrogenase and therefore macromolecular cofactors with better coenzymatic properties still must be found for the exploitat ion of these enzymes in membrane reactors.

Another way to improve use might be to isolate and test enzymes obtained from other organisms, in the hope that they will recognize the coenzyme derivatives better. This hope is based on the results of Katayama et al., a2 who found that enzymes from different sources have markedly different activities with modified NAD(H) with a few thermophilic enzymes that are even more active with macro- molecular NAD ÷ than with NAD ÷.

Acknowledgements

This work was supported by the Consiglio Nazionale delle Ricerche, Rome, Progetto Finalizzato 'Chimica Fine e Secondaria' , and by the Biotechnology Programme of the Commission of the European Communities.

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