Deamination of 2′,3′-O-isopropylideneadenosine-5′- carboxylic acid catalyzed by adenosine deaminase (ADA) and adenylate deaminase (AMPDA): Influence of substrate ionization on the activity of the enzymes

Article abstract of DOI:10.1080/15257770601052356

Adenosine deaminase (ADA) and adenylate deaminase (AMPDA) catalyze the deamination of 2′,3′-O-isopropylideneadenosine-5′-carboxylic acid to the corresponding inosine derivative and dependence of the rate of enzymatic reaction on the ionization degree of the substrate has been studied at different pH values. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770601052356

Nucleosides, Nucleotides, and Nucleic Acids, 26:121–127, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770601052356
DEAMINATION OF 2ꢀ,3ꢀ-O-ISOPROPYLIDENEADENOSINE-5ꢀ-
CARBOXYLIC ACID CATALYZED BY ADENOSINE DEAMINASE (ADA)
AND ADENYLATE DEAMINASE (AMPDA): INFLUENCE
OF SUBSTRATE IONIZATION ON THE ACTIVITY OF THE ENZYMES
Pierangela Ciuffreda and Laura Alessandrini
Dipartimento di Scienze
2
Precliniche LITA Vialba–Universita` degli Studi di Milano, Milano, Italy
Radmila Pavlovic and Enzo Santaniello Dipartimento di Medicina, Chirurgia e
2
Odontoiatria–Universita` degli Studi di Milano, Milano, Italy
Adenosine deaminase (ADA) and adenylate deaminase (AMPDA) catalyze the deamination
2
of 2 ꢁ,3 ꢁ-O-isopropylideneadenosine-5ꢁ-carboxylic acid to the corresponding inosine derivative and
dependence of the rate of enzymatic reaction on the ionization degree of the substrate has been studied
at different pH values.
Keywords 2ꢁ,3ꢁ-O-isopropylideneadenosine-5ꢁ-carboxylic acid; adenosine deaminase;
adenylate deaminase
INTRODUCTION
Adenosine deaminase (adenosine aminohydrolase, ADA, EC 3.5.4.4)
and adenylate deaminase (5ꢁ-adenylic acid deaminase, AMP deaminase, AM
PDA, EC 3.5.4.6) are metalloenzymes that belong to the class of hydrolases
and catalyze the irreversible elimination of ammonia from adenosine 1a
and adenosine 5ꢁ-phosphate (adenylic acid, AMP, 1b) to the corresponding
inosines 2a, b (Scheme 1).[1]
Both enzymes are commercially available at a considerable level of
purity and adequate activity and have been used as biocatalysts for chemo-
enzymatic transformations in nucleoside chemistry.[2] For instance, starting
from the initial observation that the steric hindrance at the positions 2ꢁ
and 3ꢁ in 2ꢁ,3ꢁ-isopropylidene adenosine 3a is well tolerated by the enzyme
Received 23 May 2006; accepted 25 September 2006.
This work has been financially supported by Universita` degli Studi di Milano (Fondi FIRST).
Address correspondence to Enzo Santaniello, Dipartimento di Medicina, Chirurgia
e Odontoiatria—Universita` degli Studi di Milano, Via A. di Rudin`ı, 8-20142, Milano, Italy. E-mail:
Enzo.Santaniello@unimi.it
121

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P. Ciuffreda et al.
SCHEME 1 ADA- and AMPDA-catalyzed deamination of adenosine (1a) and adenosine 5ꢁ-phosphate
(1b).
ADA,[3] our group has studied ADA- and AMPDA-catalyzed deamination of
a few 2ꢁ,3ꢁ-isopropylidene adenosines modified at position 5ꢁ (compounds
3b–e, Figure 1).[4,5]
The aforementioned results have shown that both enzymes can tolerate
a substitution at the 2ꢁ,3ꢁ-positions. AMPDA seems a more versatile biocata-
lyst, since it converts all the compounds 3a–e to the corresponding inosine
derivatives in a time well suitable for preparative purposes. ADA instead
requires at the 5ꢁ-position the presence of a hydroxy group for binding to
the residue of Asp-19 through a hydrogen bond, which is essential for the
catalytic mechanism.[6]
We have now investigated ADA and AMPDA activity with 2ꢁ,3ꢁ-
isopropylideneadenosine-5ꢁ-carboxylic acid (4a) where the 5ꢁ-CH2OH moi-
ety of compound 3a is substituted by a carboxylic group (Figure 2).
FIGURE 1 Structure of adenosine analogues 3a–e

2ꢁ,3ꢁ-O-Isopropylideneadenosine-5ꢁ-carboxylic Acid
123
FIGURE 2 Structure 2ꢁ,3ꢁ-isopropylideneadenosine-5ꢁ-carboxylic acid in uncharged and charged forms
(4a and 4b).
However, aqueous solutions of compound 4a should consist of charged
and uncharged species (4a and 4b) according to an equilibrium that should
be pH-dependent. Because of the essential hydrogen bond with Asp-19,
the ionization degree of carboxy group should influence ADA and AMPDA
activity. Considering a pK of 4–5 for the COOH group, compound 4a should
prevail at pH values <4–5 and the carboxylate anion in 4b should be more
present at pH >5–6. On the other hand, AMPDA catalytic performance with
compound 4a could be influenced in a different manner by pH variations,
since at the optimum value of pH 6.5, the physiological substrate 5ꢁ-adenylic
acid should be almost exclusively present in an anionic form.
Here, we report the results of our studies on the activity of ADA and
AMPDA on compound 4a at different pH values of the enzymatic reaction
medium, consisting in a 3% DMSO aqueous solution.[4]
RESULTS AND DISCUSSION
Activity of ADA and AMPDA on 2ꢁ,3ꢁ-Isopropylidene Adenosine 3a
at Different pH
It cannot be excluded that, at various pH, ionization of amino acid
residues of catalytic site or purine bases in nucleoside could influence the
enzyme activity. Therefore, experiments at different pH values were prelim-
inarily performed with both enzymes using 2ꢁ,3ꢁ-isopropylidene adenosine
3a that contained a non-ionizable group at the critical 5ꢁ-position. We have
found that, within a range of pH 6–8, ADA activity is not significantly
affected, in agreement with previously reported observations.[7] Our data
show that for this enzyme the lowest working acidity remains at pH 4 (24
hours for 20% conversion).

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P. Ciuffreda et al.
TABLE 1 Reaction time (hours for 100% conversion) of ADA and AMPDA catalyzed deamination of
substrates 3a and 4a at different pH
ADA
ADA
ADA
ADA
AMPDA AMPDA AMPDA AMPDA
Substrate
pH = 4.0 pH = 6.0 pH = 7.4 pH = 8.0 pH = 4.0 pH = 6.0 pH = 6.5 pH = 8.0
3a
4a
24a
24b
0.5
3.0
0.25
7.5
1.0
1.5
1.25
0.75
1.0
0.75
0.75
1.0
0.5
9.0a
a20% conversion.
b46% conversion.
Additionally, AMPDA showed no appreciable variation of activity within
a range going from pH 4.0 to 8.0 (Table 1). Our results suggest that the
pH-dependent ionization of enzyme aminoacid or purine basic residues
does not appreciably influence enzymatic rates of ADA and AMPDA. On
these bases, we have undertaken studies on ADA- and AMPDA-catalyzed
deamination of 2ꢁ,3ꢁ-isopropylidene adenosine 5ꢁ-carboxylic acid (4a) at
different pH values.
ADA- and AMPDA-Catalyzed Deamination of
2ꢁ,3ꢁ-Isopropylideneadenosine-5ꢁ-Carboxylic Acid (4a) at Different
pH Values
We have prepared compound (4a) from the corresponding adenosine
derivative 3a with excess KMnO4 under described conditions[8] (Scheme 2).
SCHEME 2 Synthesis of 2ꢁ,3ꢁ-isopropylideneadenosine-5ꢁ-carboxylic acid (4a) and related enzyme-
catalyzed deamination to inosine derivative 5.
Results from ADA- and AMPDA-catalyzed reactions of deamination of
2ꢁ,3ꢁ-isopropylideneadenosine-5ꢁ-carboxylic acid (4a) at different pH values
are shown in Table 1. At the optimum pH value 7.4, ADA-catalyzed deam-
ination proceeded quantitatively in 7.5 hours, a reaction rate considerably
lower than the corresponding adenosine isopropylidene derivative 3a (0.25
hour). Interestingly, at slightly higher value (pH 8.0) the reaction rate was
further lowered (20% conversion in 9 hours) while it was relatively higher

2ꢁ,3ꢁ-O-Isopropylideneadenosine-5ꢁ-carboxylic Acid
125
(3 hours) at pH 6.0. Although pH 4.0 is the lowest acceptable limit for ADA
activity, it is interesting to observe that a significant 46% conversion was
reached in 24 hours for 2ꢁ,3ꢁ-isopropylidene adenosine 5ꢁ-carboxylic acid
(4a) (within the same time, 20% conversion of 3a was obtained). These
results clearly indicate that for ADA activity the presence of a negative
charge at the critical position 5ꢁ has a detrimental effect on the rate of
the enzymatic reaction. Considering the active site described for ADA,[6]
the catalytically crucial hydrogen bond between Asp-19 and the 5ꢁ-hydroxyl
group is substituted by repulsion between Asp-19 and the 5ꢁ-carboxylate
anion of 4b. At more acidic pH the equilibrium is shifted towards the neutral
form of the 5ꢁ-carboxylic group and compound 4a is transformed by the
enzyme, although at a lower rate than 3a.
Compared to ADA, AMPDA activity with substrate 4a is not significantly
affected by pH values higher than the optimal 6.5 and is only slightly lowered
at pH 4. Interestingly, AMPDA is more active than ADA at low pH values,
although its activity with physiological substrate 5ꢁ-adenylate anion indicates
a preference for ionized substrate 4b.
EXPERIMENTAL
General
Melting points were recorded on SMP3 instrument (Stuart Scientific,
UK) and are uncorrected. The NMR spectra were recorded on a Bruker
AVANCE 500 spectrometer operating at 500.13 and 125.76 MHz for 1H and
13C. The central peak of DMSO-d6 signals (2.49 ppm for 1H and 39.50 ppm
for 13C) was used as internal standard. The chemical shifts are reported in
parts per million and coupling constants (J ) are given in Hertz. Mass spectra
were recorded on Finnigan LCQ-Deca (Termoquest) in ESI negative-ion
mode, KV 5.00, 225◦C, 15 V. HPLC analyses were carried out on a Jasco
HPLC instrument with an Uvidec 100 II UV detector operating at 260 nm
using an Alltech Hypersil BDS C18 (4.6 mm × 250 mm). The eluant was
phosphate buffer at pH 6.0 containing 20% or 25% acetonitrile for 3a and
4a, respectively, at a 1 ml/minute flow rate.
Enzymes were obtained from Sigma, specifically ADA from calf intestinal
mucosa (type II, 2.2 units/mg protein) and AMPDA from Aspergillus species
(0.107 units/mg protein). 2ꢁ,3ꢁ-O-isopropylideneadenosine 3a was prepared
according to a published procedure.[9] 2ꢁ,3ꢁ-O-isopropylideneadenosine-5ꢁ-
carboxylic acid (4a) was obtained from the corresponding adenosine deriva-
tive 3a with excess KMnO4 under described conditions:[8] mp 244–246◦C
1
(dec) (lit.[10] 246–249◦C dec); H NMR (DMSO-d6) δ 8.25 (1H, s, H-8),
8.08 (1H, s, H-2), 7.35 (2H, s, NH2), 6.32 (1H, bs, J = <1.0 Hz, H-1ꢁ),
5.52 (1H, dd, J = 2.0, 6.0 Hz, H-3ꢁ), 5.45 (1H, d, J = <1.0, 6.0 Hz, H-2ꢁ),
4.68 (1H, d, J = 2.0 Hz, H-4ꢁ), 1.50 (3H, s, CCH3), 1.33 (3H, s, CCH3).

126
P. Ciuffreda et al.
13C NMR (DMSO-d6) δ 171.17 (COOH), 156.36 (C-6), 152.74 (C-2), 149.58
(C-4), 140.91 (C-8), 119.18 (C-5), 113.18 (CCH3), 89.99 (C-1ꢁ), 85.88 (C-3ꢁ),
84.22 (C-2ꢁ), 83.85 (C-4ꢁ), 26.92 (CCH3), 25.34 (CCH3). M/z 320 (M-1)−,
663 [(M-1)+(M-1)+Na]−.
Enzymatic Deamination of Adenosine Derivatives 3a and 4a
Compounds 3a and 4a (20 mg) in phosphate buffer at different pH
(50 mM, 10 mL) containing 3% DMSO were treated with ADA (2 mg)
or AMPDA (20 mg) for the time indicated in Table 1. The progress of
reactions was monitored by HPLC, as previously indicated. The solution
was lyophilized and the residue crystallized from methanol as white solids.
2ꢁ,3ꢁ-O-isopropylideneinosine showed physical characteristics in agreement
with published data.[11]
[12]
2ꢁ,3ꢁ-O-isopropylideneinosine-5ꢁ-carboxylic acid
(5): mp 252–254◦C
(dec) (lit.[12] 252◦C); 1H NMR (DMSO-d6) δ 8.45 (1H, s, H-8), 8.01 (1H, s,
H-2), 6.18 (1H, bs, J = <1.0 Hz, H-1ꢁ), 5.21–5.18 (2H, m, H-2ꢁ and H-3ꢁ),
4.49 (1H, d, J = 2.0 Hz, H-4ꢁ), 1.49 (3H, s, CCH3), 1.29 (3H, s, CCH3).
13C NMR (DMSO-d6) δ 172.14 (COOH), 157.12 (C-6), 148.45 (C-4), 146.01
(C-2), 140.33 (C-8), 124.28 (C-5), 113.16 (CCH3), 90.61 (C-1ꢁ), 87.09 (C-3ꢁ),
84.66 (C-2ꢁ), 84.30 (C-4ꢁ), 27.09 (CCH3), 25.31 (CCH3). M/z 321 (M -1)−,
665 [(M-1)+(M-1)+Na]−.
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