Mechanistic diagnoses of N-ribohydrolases and purine nucleoside phosphorylase

Full text of DOI:10.1021/ja953537z

J. Am. Chem. Soc. 1996, 118, 2111-2112
2111
(3) ionization of the 2′-hydroxyl to stabilize the developing
oxycarbonium, as proposed for base-catalyzed NAD+ solvolysis
and established in chemical models and in calf spleen NAD+
glycohydrolase.12 A combination of these features may also
occur in some N-ribohydrolases.
Mechanistic Diagnoses of N-Ribohydrolases and
Purine Nucleoside Phosphorylase
L. John Mazzella,† David W. Parkin,† Peter C. Tyler,‡
Richard H. Furneaux,‡ and Vern L. Schramm*,†
We report here that p-nitrophenyl â-D-ribofuranoside (nitro-
phenylriboside) and its 5-phosphate can be used as substrates
to distinguish these mechanisms for five members of the family
of N-ribohydrolases. The favorable p-nitrophenyl leaving group
lacks the pyrimidine and purine ring nitrogens which are proton
acceptors in acid-catalyzed solvolysis. If substrate activation
required protonation (or hydrogen bonds of similar electron-
withdrawing strength) at the nitrogen(s) of the leaving group,
nitrophenylriboside would be a poor substrate. In contrast,
substantial enzymatic activity with this compound would
indicate mechanisms in which the enzyme interacts with the
ribosyl to stabilize an oxycarbonium-ion transition state, or
ionizes a ribosyl hydroxyl to facilitate the unassisted departure
of the p-nitrophenolate ion,12d or protonates the â-oxygen bridge
to the p-nitrophenyl group,12c These mechanistic proposals can
be further distinguished by comparing the kinetic parameters
for enzyme-catalyzed hydrolysis of the normal substrate and
nitrophenylriboside as a function of pH.
D-Ribose (I) was converted to p-nitrophenyl â-D-ribofurano-
side (III) as previously described13,14 and phosphorylated at the
5-position (Scheme 1). Kinetic parameters for hydrolysis of
ribosides III or V by several enzymes are compared with those
of normal substrates in Table 1. The enzymes with the most
stringent substrate specificities, GI-nucleoside hydrolase, AMP
nucleosidase, and purine nucleoside phosphorylase, gave kcat/
Km values with nitrophenylriboside (or its 5-phosphate) which
are 4 × 10-5, 3 × 10-7, and 1 × 10-6 of those for the substrates,
guanosine, AMP, and inosine, respectively. In contrast, the
same comparison of kcat/Km values for the nonspecific IU-
nucleoside hydrolase favored III by a factor of 54. The ribosyl
moiety is unchanged between the normal substrates and the
nitrophenylribosides. The Km values for nitrophenylribosides
are within a factor of 42 of those for the normal substrates for
all of the enzymes in Table 1, implicating the ribosyl in substrate
recognition. Catalysis primarily by ribose diol anion formation12d
or by enforcing ribo-oxycarbonium formation5a would give good
activity with nitrophenylriboside; therefore, the enzymes with
stringent base specificity are likely to involve a substantive
component of leaving-group activation. Protonation at N7 of
AMP is known to be an important feature of the transition states
stabilized by AMP nucleosidase, since formycin 5′-phosphate,
which is protonated at this position, is a transition state
Department of Biochemistry
Albert Einstein College of Medicine
Bronx, New York 10461,
Industrial Research Limited
P.O. Box 31-310, Lower Hutt, New Zealand
ReceiVed October 23, 1995
The N-ribohydrolases and transferases represent a class of
catalytic activities of increased interest, since they are involved
in novel pathways of purine salvage in protozoan parasites,1 in
nucleic acid repair,2 in the actions of bacterial and plant toxins,3
and in regulation of calcium ion flux.4 Transition states for
purine N-ribohydrolases and phosphorylases share ribosyl
oxycarbonium-ion character.5 Substrate specificity is conferred
by the nature of the enzyme-activated nucleophile and the
leaving-group interactions. Among the known N-ribohydro-
lases, specificity is high for the ribosyl group but varies for the
leaving group purine or pyrimidine. The inosine-uridine
nucleoside hydrolase (IU-nucleoside hydrolase) from the try-
panosome Crithidia fasciculata hydrolyzes all of the naturally
occurring purine and pyrimidine ribonucleosides with similar
catalytic efficiencies, while the guanosine-inosine enzyme (GI-
nucleoside hydrolase) from the same organism has a strong
preference for the eponymous substrates and is nearly inert with
other purine and pyrimidine nucleosides.6 A nucleoside hy-
drolase from Trypanosoma brucei brucei demonstrates purine
nucleoside specificity but includes inosine, adenosine, and
guanosine as good substrates (IAG-nucleoside hydrolase).7 AMP
nucleosidase from bacterial sources is highly specific for the
adenine base, and the 5′-phosphoryl is required for hydrolysis
of the N-ribosidic bond.8 Purine nucleoside phosphorylase from
mammalian sources is specific for inosine and guanosine as
substrates and activates phosphate or arsenate anions to attack
C1′ of these nucleosides.9
The common catalytic feature of these enzymes is the
oxycarbonium-ion character of the transition state, in which
cleavage of the C-N ribosidic bond occurs by an SN1-like
mechanism. This transition state can be achieved by three
distinct reaction pathways: (1) activation of the leaving group,
as in the case for the acid-catalyzed solvolysis of purine
nucleosides;10 (2) catalytic site interactions with the ribosyl
moiety, to stabilize both the oxycarbonium-ion charge and
geometry similar to mechanisms proposed for lysozyme;11 and
(10) (a) Parkin, D. W.; Leung, H. B.; Schramm, V. L. J. Biol. Chem.
1984, 259, 9411. (b) Garrett, E. R.; Mehta, P. J. J. Am. Chem. Soc. 1972,
94, 8532.
(11) Imoto, T.; Johnson, L. M.; North, A. C. T.; Phillips, D. C.; Rupley,
J. A. In The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press: New
York, 1972; Vol. 7, p 665.
(12) (a) Oppenheimer, N. J.; Handlon, A. L. in The Enzymes, 3rd ed.;
Sigman, D. S., Ed.; Academic Press: San Diego, 1992; Vol. 20, p 454. (b)
Cherian, X. M.; VanArman, S. A.; Czarnik, A. W. J. Am. Chem. Soc. 1990,
112, 4490. (c) Rosenberg, S.; Kirsch, J. F. Biochemistry 1981, 20, 3196.
(d) Handlon, A. L.; Xu, C.; Muller-Steffner, H. M.; Schuber, F.; Oppen-
heimer, N. J. J. Am. Chem. Soc. 1994, 116, 12087.
* Corresponding author: telephone, (718) 430-2813; FAX, (718) 892-
0703; e-mail, vern@aecom.yu.edu.
† Albert Einstein College of Medicine.
‡ Industrial Research Limited.
(1) Hammond, D. J.; Gutteridge, W. E. Mol. Biochem. Parasitol. 1984,
13, 243.
(2) Sancar, A.; Sancar, G. B. Annu. ReV. Biochem. 1988, 57, 29.
(3) (a) Endo, Y.; Gluck, A.; Wool, I. G. J. Mol. Biol. 1991, 221, 193.
(b) Aktories, K., Ed. Current Topics in Microbiology and Immunology. ADP-
Ribosylating Toxins; Springer-Verlag: Berlin, 1992.
(4) Lee, H. C.; Galione, A.; Walseth, T. F. Vitam. Horm. 1994, 48, 199.
(5) (a) Mentch, F.; Parkin, D. W.; Schramm, V. L. Biochemistry 1987,
26, 921. (b) Horenstein, B. A.; Parkin, D. W.; Estupinan, B.; Schramm, V.
L. Biochemistry 1991, 30, 10788. (c) Kline, P. C.; Schramm, V. L.
Biochemistry 1995, 34, 1153.
(6) (a) Parkin, D. W.; Horenstein, B. A.; Abdulah, D. R.; Estupinan, B.;
Schramm, V. L. J. Biol. Chem. 1991, 266, 20658. (b) Estupin˜a´n, B.;
Schramm, V. L. J. Biol. Chem. 1994, 269, 23068.
(7) Parkin, D. W. Unpublished observation, manuscript in preparation.
(8) (a) Hurwitz, J.; Heppel, L. A.; Horecker, B. L. J. Biol. Chem. 1957,
226, 525. (b) DeWolf, W. E., Jr.; Fullin, F. A.; Schramm, V. L. J. Biol.
Chem. 1979, 254, 10868.
(9) (a) Kline, P. C.; Schramm, V. L. Biochemistry 1992, 31, 5964. (b)
Kline, P. C.; Schramm, V. L. Biochemistry 1993, 32, 13212.
(13) Recondo, E. F.; Rinderknecht, H. HelV. Chim. Acta 1959, 42, 1171.
(14) Honma, K.; Nakazima, K.; Uematsu, T.; Hamada, A. Chem. Pharm.
Bull. 1976, 24, 394. 1H NMR of III, (DMSO-d6): δ 8.20 and 7.17 (2H
each, d, Ar), 5.63 (1H, d, J ) 0.7 Hz, H-1), 4.07-4.02 (2H, m, H-2,3),
3.96-3.92 (1H, m, H-4), 3.57-3.50 (1H, m, H-5), 3.37-3.29 (1H, m, H-5′).
13C NMR: δ 163.1, 142.7, 127.0, 117.9 (Ar), 106.4 (C-1), 86.4 (C-4), 75.9,
71.6 (C-2,3), 63.6 (C-5). Yield of IV from III was 20%, and yield for
1
conversion of IV to V was 56%. H NMR of V (D2O): δ 8.12 and 7.08
(2H each, d, Ar), 5.71 (1H, s, H-1), 4.43-4.11 (3H, m), 3.97-3.70 (2H,
m). 13C NMR: δ 164.1, 144.9, 128.9, 119.3 (Ar), 107.6 (C-1), 86.1 (Jc,p
8.2 Hz, C-4), 77.3, 73.9 (C-2,3), 67.7 (Jc,p ) 4.6 Hz, C-5).
)
(15) (a) Leung, H. B.; Kvalnes-Krick, K. L.; Myer, S. L.; deRiel, J. K.;
Schramm, V. L. Biochemistry 1989, 28, 8726. (b) Leung, H. B.; Schramm,
V. L. J. Biol. Chem. 1980, 255, 10867.
0002-7863/96/1518-2111$12.00/0 © 1996 American Chemical Society

2112 J. Am. Chem. Soc., Vol. 118, No. 8, 1996
Table 1. Kinetic Parameters for N-Ribohydrolases and Purine Nucleoside Phosphorylase with Nucleosides and Nitrophenyl Ribosidea
nitrophenyl riboside substrate
purine substrateb
Communications to the Editor
k
cat/Km
k
cat/Km
k
cat/Km
enzyme
kcat (s-1
)
Km (µM)
(M-1 s-1
)
substrate kcat (s-1
)
Km (µM) (M-1 s-1
)
ratiog
IU-nucleoside hydrolaseR,c
IAG-nucleoside hydrolaseR,d
GI-nucleoside hydrolaseR,c
AMP nucleosidaseP,e
239 ( 32
58 ( 23
4.1 × 106 inosine
1.5 × 103 inosine
1.4 × 102 guanosine
8.9 × 10-1 inosine
28
34
231
27
380
18
77
150
19
7.6 × 104 54
0.82 ( 0.03
0.07 ( 0.01
560 (50
1.9 × 106 8 × 10-4
3.2 × 106 4 × 10-5
1.8 × 105 3 × 10-7
6.3 × 105 1 × 10-6
468 ( 130
0.0004 ( 0.0001
6250 ( 1700 6.2 × 10-2 AMP
purine nucleoside phosphorylaseR,f 0.00020 ( 0.00004 224 ( 76
12
a The enzymes were highly purified samples from C. fasciculata,c,6 T. brucei brucei,d,18 Escherichia coli,e,15 and bovine spleen.f,9a Assays were
at pH 8.0 in 50 mM HEPES, 30 °C. For the allosteric AMP nucleosidase, MgATP was present at 100 µM. Purine nucleoside phosphorylase was
assayed for phosphorolysis by including 3 mM phosphate in assay mixtures. The superscripts R and P refer to nitrophenyl riboside and nitrophenyl
riboside 5-phosphate, respectively, as substrates. b Substrate specificity, for purine substrates, the kinetic constants, and their standard errors are
available in refs 6a,b, 7, 15b, and 9. g The kcat/Km ratio compares the kcat/Km for nitrophenyl riboside to that for the indicated purine substrate.
Scheme 1. Synthesis of Nitrophenylribose (III) and
Nitrophenylribose 5-Phosphate (V)a
Figure 1. Hydrolysis of nitrophenylriboside and inosine by IU-
nucleoside hydrolase as a function of pH. The data for inosine is
a Reagents: i, p-nitrophenol, BF3‚OEt2; ii, NaOH, MeOH; iii, N,N-
replotted from ref 18. The data for hydrolysis of nitrophenylriboside
was fitted to the equation log Vmax ) log[(V1+V2(Ka/[H+]))/(1+Ka/
diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine, tetrazole, then
[H+])].
MCPBA; iv, H2, Pd/C, EtOH, then NaOH.
analogue.8b,16 Tight binding of hypoxanthine by purine nucleo-
side phosphorylase9a and N7 protonation deduced from transition
state analysis5c also support leaving-group activation.
hydrolase are not required for the facile solvolysis of nitrophe-
nylriboside. Solvolysis is therefore proposed to occur primarily
through activation of the ribosyl toward the oxycarbonium ion.
The oxycarbonium-stabilizing group provides weak (3-fold)
catalytic assistance, but the leaving-group proton donor is not
required for solvolysis of the nitrophenylriboside. The enzymes
which are poor catalysts for this substrate have mechanisms
which differ, despite similarities in the nature of the ribosyl at
the transition state. Activation of the leaving group by proton-
ation and/or other electrostatic interactions contributes more
significantly in lowering the transition state barrier for the
enzymes which are inefficient in solvolysis of nitrophenyl-
riboside.
Nitrophenylriboside, like its hexose counterparts,12b,17 under-
goes specific acid- and base-catalyzed solvolysis. The riboside
is relatively stable from pH 4 to 10 pH with the solvolytic rate
increasing rapidly at more extreme pH values, with a near-
symmetric response to acid and base. Acid-catalyzed O-
glycoside solvolysis occurs via protonation of the glycosidic
oxygen while base-catalyzed solvolysis is thought to occur via
internal attack of ionized O2 on the anomeric carbon.12b These
protonic interactions can be detected by the pH dependence of
Vmax
.
Nitrophenylribosides are mechanistic tools to identify en-
zymes which catalyze N-riboside hydrolysis by forming the ribo-
oxycarbonium ion. The extent of leaving-group activation can
then be evaluated. In the test group of five N-ribosidases, the
nonspecific IU-nucleoside hydrolase is most efficient in ribo-
oxycarbonium-ion formation to achieve the transition state.
Leaving-group activation plays more important roles for the
other enzymes. The (1.8 × 108)-fold range in catalytic
efficiencies (substrate/nitrophenylriboside) for these enzymes
indicates considerable mechanistic diversity among the N-
ribosidases.
Hydrolysis of inosine by IU-nucleoside hydrolase requires
one proton donor, pKa 9.1, and one proton acceptor, pKa 7.1
(Figure 1). These groups are proposed to stabilize the ribo-
oxycarbonium ion formed at the transition state and assist the
leaving group by N7 protonation.18 Hydrolysis of nitrophenyl-
riboside under the same conditions reveals no acid or base
groups in the enzyme that are essential for catalysis. Acid- and/
or base-catalyzed solvolysis would be expected to show an
essential group at pH extremes, with unit slope(s). Instead, the
group (pKa 6.8) which has been proposed to stabilize the
oxycarbonium is shown to cause a 3-fold increase in reaction
rate but is not essential.
Acknowledgment. The work was supported by the National
Institutes of Health, the United States Army Medical Research and
Development Command under Contract No. DAMD 17-93-C-3051, and
the New Zealand Foundation for Research, Science and Technology.
The views, opinions, and/or findings contained in this report are those
of the authors and should not be construed as an official Department
of Army position, policy, or decision unless so designated by other
documentation.
The results demonstrate that the ionizable groups which are
required for acid-base solvolysis of inosine by IU-nucleoside
(16) (a) Ehrlich, J.; Schramm, V. L. Biochemistry 1994, 33, 8890. (b)
Giranda, V. L.; Berman, H. M.; Schramm, V. L. Biochemistry 1988, 27,
5813.
(17) (a) Snyder, J. A.; Link, K. P. J. Am. Chem. Soc. 1952, 74, 1883.
(b) Sinnott, M. L.; Jencks, W. P. Ibid. 1980, 102, 2026.
(18) Parkin, D. W.; Schramm, V. L. Biochemistry 1995, 34, 13961.
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