Ribocation Transition State Capture and Rebound in Human Purine Nucleoside Phosphorylase

Article abstract of DOI:10.1016/j.chembiol.2009.07.012

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and α-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state

Full text of DOI:10.1016/j.chembiol.2009.07.012

Chemistry & Biology
Article
Ribocation Transition State Capture and Rebound
in Human Purine Nucleoside Phosphorylase
Mahmoud Ghanem,1,2,3,4 Andrew S. Murkin,1,3,5 and Vern L. Schramm1,
*
1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA
2Faculty of Veterinary Medicine, Cairo University, Giza, Egypt
3These authors contributed equally to this work
4Present address: Eli Lilly Pharmaceuticals, Inc., Indianapolis, IN 46285, USA
5Present address: Department of Chemistry, University at Buffalo, SUNY, Buffalo, NY 14260, USA
*Correspondence: vern@aecom.yu.edu
DOI 10.1016/j.chembiol.2009.07.012
˚
SUMMARY
ꢁ1.8 A (Kline and Schramm, 1993). Bovine PNP catalyzes the
slow hydrolysis of inosine to hypoxanthine and ribose in the
Purine nucleoside phosphorylase (PNP) catalyzes absence of phosphate with a burst of 2 3 10ꢀ3 Sꢀ1 turnover
number of 1.3 3 10ꢀ4 sꢀ1 (Figure 1, path c) (Kline and Schramm,
1992, 1995). Inosine hydrolysis has not been previously reported
the phosphorolysis of 6-oxy-purine nucleosides to
the corresponding purine base and a-D-ribose
1-phosphate. Its genetic loss causes a lethal T cell
deficiency. The highly reactive ribocation transition
state of human PNP is protected from solvent by
hydrophobic residues that sequester the catalytic
site. The catalytic site was enlarged by replacing indi-
for human PNP.
Crystal structures of human PNP complexed with substrate
and TS analogs have defined the active site residues in contact
with the purine nucleoside and the phosphate nucleophile (de
Azevedo, et al., 2003; Koellner, et al., 1997; Rinaldo-Matthis,
et al., 2008; Shi, et al., 2004). Human PNP is a homotrimer,
vidual catalytic site amino acids with glycine. Reac-
tivity of the ribocation transition state was tested
for capture by water and other nucleophiles. In the
and the catalytic site of PNP is located near the subunit-subunit
interfaces. The catalytic site conforms closely to bound inosine
with residues N243, E201, H257, F200, Y88, M219, and F1590
absence of phosphate, inosine is hydrolyzed by (from the adjacent subunit) as contacts for the nucleoside. The
side chains of N243 and E201 are involved in leaving-group acti-
native, Y88G, F159G, H257G, and F200G enzymes.
vation by forming hydrogen-bond contacts with the purine base
(N7H and N1H, respectively) (Ghanem, et al., 2008b; Nunez,
et al., 2006). Residues F1590, H257, F200, and Y88 form a hydro-
phobic cover to the catalytic site and are proposed to shield
reactants in the catalytic site from bulk solvent (Figure 2)
Phosphorolysis but not hydrolysis is detected when
phosphate is bound. An unprecedented N9-to-N3
isomerization of inosine is catalyzed by H257G and
F200G in the presence of phosphate and by all
PNPs in the absence of phosphate. These results
(Saen-Oon, et al., 2008a). The phenyl group of F200 is perpen-
establish a ribocation lifetime too short to permit
capture by water. An enlarged catalytic site permits
ribocation formation with relaxed geometric con-
straints, permitting nucleophilic rebound and N3-
inosine isomerization.
dicular to the purine base, and residues Y88, F1590, H257 and
the bound phosphate nucleophile provide contacts to the ribose.
Residue F1590 from a mobile loop in the adjacent subunit covers
the top surface of the ribosyl group of the purine nucleoside
(Figure 2). Quantum mechanical simulations and laser-induced
temperature-jump studies establish that dynamic motion of
this residue is closely associated with TS formation (Ghanem,
et al., 2009; Saen-Oon, et al., 2008a). H257 is a key residue in
positioning the ‘‘oxygen stack’’ (H257:NdꢀO50ꢀO40ꢀOP) that
INTRODUCTION
Purine nucleoside phosphorylase (PNP) catalyzes the reversible contributes to catalysis through a vibrational promoting mode
phosphorolysis of 6-oxo-purine (deoxy)ribonucleosides to the of the three oxygen atoms to provide electron density changes
corresponding purine base and a-D-ribose 1-phosphate (Fig- to destabilize the ribosyl group, form the carbocation TS and
ure 1, path b) (Schramm, 2005; Stoeckler, et al., 1978, 1980). thereby enhance departure of the purine-base leaving group
This enzyme is a current target for clinical trials involving T cell (Murkin, et al., 2007; Saen-Oon, et al., 2008a). The crystal struc-
cancers and autoimmune disease (BioCryst, 2009). Kinetic ture of human PNP in complex with a TS analog (Immucillin-H)
isotope effects have characterized the reaction catalyzed by and phosphate shows structural water molecules near O6
˚
˚
human PNP as proceeding via a fully dissociated ribocation tran- (2.6 A) and another within 4.4 A of the ribosyl anomeric carbon
sition state (TS) with complete leaving-group departure prior to (Figure 2), and these structural waters are also found in the cata-
ribosyl migration to the phosphate nucleophile (Lewandowicz lytic site of bovine PNP (Fedorov, et al., 2001).
and Schramm, 2004). Despite the high amino acid sequence We explored two possibilities for water capture of the highly
identity (87%) with human PNP, bovine PNP forms an earlier reactive ribocation: (1) water molecules located within the cata-
ribooxacarbenium-ion TS featuring a C10ꢀN9 bond length of lytic site prior to ribocation formation, or (2) water molecules that
Chemistry & Biology 16, 971–979, September 25, 2009 ª2009 Elsevier Ltd All rights reserved 971

Chemistry & Biology
Ribocation Capture in Human PNP
Figure 1. Reactions Catalyzed by Purine Nucleoside Phosphorylase
Depending on reaction conditions, the ribo-oxacarbenium-ion transition state can partition between (a) return to inosine [1], (b) reaction with phosphate to yield
hypoxanthine [2] and a-D-ribose 1-phosphate [4], (c) reaction with water to yield hypoxanthine [2] and D-ribose [3], or (d) reaction with N3 of hypoxanthine to
yield N3-isoinosine [5].
diffuse from bulk solvent into the catalytic site as a consequence tions with [10-13C]inosine as substrate. Native PNP and cata-
of catalytic-site mutations. Water capture of the ribocation in the lytic-site glycine mutants of human PNP were analyzed. Four
presence of phosphate would partition the pentose between different glycine mutants of residues that are proposed to shield
ribose and a-D-ribose 1-phosphate products. This approach the catalytic site from bulk solvent were engineered in attempts
provides a test of ribocation reactivity, its lifetime relative to to introduce a solvent leak. Partition of products between
water diffusion, and allows an estimate of the relative proclivity D-ribose and a-D-ribose 1-phosphate permit estimation of ribo-
of the ribocation to react with water, phosphate, the hypoxan- cation lifetime, based on the diffusion rate of water and the
thine leaving group or adventitious catalytic site nucleophiles. distance of the ribocation from bulk solvent in a leaky catalytic
This work contributes substantially to our understanding of the site.
ribocation transition state.
13C and 1H nuclear magnetic resonance (NMR) spectroscopy
were used to monitor the hydrolysis and phosphorolysis reac-
RESULTS
Steady-State Kinetic Properties
The steady-state kinetic parameters of four catalytic-site glycine
mutants were determined with inosine as substrate and com-
pared with those for the native enzyme. All catalytic-site glycine
mutants showed a decrease in their kcat values ranging from
2-fold (Y88G) to 40-fold (F200G) compared with native PNP
(see Table S1 available online). The catalytic efficiencies (kcat/Km)
of those mutants were reduced by ꢁ8-fold (Y88G) and up to
11,000-fold (F200G) relative to native PNP (Table S1). Purified
PNPs showed sufficient catalytic stability in the reaction
mixtures at 25ꢂC to permit collection of 13C or 1H NMR spectra
of actively equilibrating enzyme mixtures for 24 hr.
13C and 1H NMR Spectroscopy and Assignments
The 13C and 1H NMR spectra of reaction mixtures containing
PNPs (native, Y88G, F159G, H257G and F200G) with [10-13C]ino-
Figure 2. Views of the Subunit-Subunit Interface of Human PNP in
Complex with a TS Analog and Phosphate (PO4)
sine in the presence or absence of phosphate were acquired
(A) PNP catalytic-site contact residues surrounding Immucillin-H (ImmH) (PDB
and compared with the reference spectra of free [10-13C]inosine
1RR6). Adjacent subunits in the trimeric protein have been colored yellow and
and [1-13C]ribose (Figure 3 and Table S2). The 13C NMR spec-
trum of free [10-13C]inosine [1] is a singlet with a chemical shift
of 88.68 ppm (Chenon, et al., 1975a; Ebrahimi, et al., 2001).
blue.
˚
(B) Contact residues map, with distances in A units. The two water molecules
(W) closest to ImmH have been included.
972 Chemistry & Biology 16, 971–979, September 25, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
Ribocation Capture in Human PNP
Figure 3. 13C NMR (I) and 1H NMR (II) Spectra of
[10-13C]inosine, [1-13C]Ribose, and Hypoxanthine
Spectra are of (A) 5.3 mM free [10-13C]Inosine and (B)
[1-13C]ribose and hypoxanthine obtained through the irre-
versible arsenolysis of 5.3 mM [10-13C]Inosine in the pres-
ence of human PNP and 50 mM Na2HAsO4 (pH 7.4).
Upfield signals have been omitted to highlight the down-
field regions of interest. All spectra were acquired at
25ꢂC in 10% D2O. [1] Ino, inosine; [2] Hx, hypoxanthine.
This chemical shift does not match those for
ribose isomers (Figure 3B). The time course
(0–24 hr) 1H NMR spectra of mixtures of PNPs
(native, F159G, Y88G, H257G, and F200G)
with [10-13C]inosine were also acquired. The 1H
NMR spectra of [10-13C]inosine with native
PNP showed two new downfield singlets with
chemical shifts of 8.55 and 8.18 ppm and
another doublet of doublets with a chemical
shift of 6.28 ppm between ꢁ3–6 and 24 hr
(Figure 4-II). The chemical shifts resemble those
reported for the chemically synthesized N3-
isomer of inosine (3-b-D-ribofuranosyl hypoxan-
thine or N3-isoinosine) [5] (s d 8.67 or 8.59, s d
8.32 or 8.25 and d d 5.98 or 5.87 for H-8, H-2
and H-10, respectively, in d6, DMSO) (Lehikoi-
nen, et al., 1989; Tindall, et al., 1972). The rate
of isomerization was approximately 1 3 10ꢀ3
sꢀ1 (Table 1). In comparison, the four catalytic-
site glycine mutants did not form any detectable
The downfield region of the 1H NMR spectrum of free [10-13C]ino- isomerized product during the time course of 1H NMR observa-
sine shows two singlets for H8 and H2 with chemical shifts of 8.33 tion (Figure 4-III, as an example for F200G).
and 8.21 ppm, respectively, and a doublet of doublets for H-10
The identity of the isomerized product as N3-isoinosine was
0
0
(J1 ,2 = 5 Hz, JH,C = 170 Hz), with a chemical shift of 6.07 ppm determined by several independent criteria. Resolution from in-
(Figure 3A and Table S2). The peak assignments were based on osine and hypoxanthine by high-performance liquid chromatog-
the previously reported chemical shifts for purine nucleosides raphy (HPLC) established
a distinct chemical entity. The
(Chenon, et al., 1975a, 1975b). The 13C NMR spectrum of electrospray ionization mass spectrometry (ESI-MS) mass value
[1-13C]ribose [3], obtained through the irreversible arsenolysis (m/z = 270, M+H+ for the 10-13C substituted species) corre-
of [10-13C]inosine in the presence of PNP (Kline and Schramm, sponds to [10-13C]N3-isoinosine (but does not distinguish it from
1993), shows four singlets with chemical shifts of 93.94, 94.22, [10-13C]inosine or [10-13C]N7-isoinosine). UV-spectral properties
96.68 and 101.35 ppm (Figure 3B and Table S2), corresponding at pH 1, 7, and 11 matched published findings for N3-isoinosine
to the four major isomers of D-ribose. The chemical shifts and and are distinct from those reported for inosine and N7-isoino-
ratio of the four isomer singlets agree with previously determined sine (Table S3) (Lehikoinen, et al., 1989; Montgomery and
values of 95.1 (20%), 95.4 (60%), 97.8 (7%), and 102.5 ppm (13%) Thomas, 1969; Tindall, et al., 1972; Wolfenden, et al., 1966).
1
for a-D-ribopyranose, b-D-ribopyranose, a-D-ribofuranose and The H NMR spectrum of this isolated product agreed with the
b-D-ribofuranose, respectively (Kenneth et al., 1998; Ryu, et al., published chemical shift data for authentic N3-isoinosine but
2004). The 1H NMR spectrum of free hypoxanthine [2] obtained not for inosine or N7-isoinosine (Table S3) (Chenon, et al.,
through the irreversible arsenolysis of labeled inosine shows 1975a; Lehikoinen, et al., 1989; Tindall, et al., 1972). The 10-13C
two downfield singlets for H-8 and H-2, with chemical shifts of chemical shift is consistent with [10-13C]N3-isoinosine but not
8.19 and 8.17 ppm, respectively (Figure 3B).
inosine or N7-isoinosine (Table S3) (Chenon, et al., 1975a).
Phosphate-free PNP Reactions and Inosine
Isomerization
PNP Reaction Fidelity with Phosphate
Conditions were selected to permit 2 3 107 catalytic turnovers of
In the absence of phosphate, the time course (to 24 hr) 13C NMR PNP at chemical equilibrium in the presence of phosphate and
spectra of the inosine reaction mixtures for F159G, Y88G, inosine. The 13C and 1H NMR spectra of reaction mixtures with
H257G and F200G showed no spectral changes (data not native, Y88G and F159G PNPs showed only the formation of
shown). Native PNP with [10-13C]inosine showed the formation hypoxanthine [2] and a-D-[1-13C]ribose 1-phosphate [4] (Fig-
of a new singlet with a chemical shift of 90.59 ppm (Figure 4-I). ure 5). The 13C and 1H NMR spectra for H257G and F200G
Chemistry & Biology 16, 971–979, September 25, 2009 ª2009 Elsevier Ltd All rights reserved 973

Chemistry & Biology
Ribocation Capture in Human PNP
Table 1. Comparison of the N3 Isomerization and Single-
Turnover Hydrolysis Reactions for PNPs with Inosine as
Substrate
Isomerization Kineticsa
Hydrolysis Kineticsb
k
iso (sꢀ1), no
kiso (sꢀ1), with
phosphatec
PNP
phosphatec
khyd (sꢀ1 c
)
BtPNPd
Native
Y88G
Not determined
1 3 10ꢀ3
Undetectablee
Undetectablee
Undetectablee
Undetectablee
Not determined
Undetectablee
Undetectablee
Undetectablee
7.0 3 10ꢀ5
2.0 3 10ꢀ3
(7.5 1.6) 3 10ꢀ4
(1.7 0.5) 3 10ꢀ3
(3.0 0.8) 3 10ꢀ3
(4.8 0.4) 3 10ꢀ4
(3.6 1.2) 3 10ꢀ3
F159G
H257G
F200G
4.2 3 10ꢀ4
a Isomerization assays were performed with 5.3 mM inosine in 20 mM
Tris-HCl (pH 7.4) in the presence or absence of 50 mM KH2PO4
(pH 7.4). PNPs were added to give a phosphorolysis Vmax of 2.6 3
10ꢀ4 M sꢀ1 (kcat 3 [E]T).
b Hydrolysis assays were performed with equal amounts of inosine and
PNP (80 mM) in 20 mM Tris-Cl (pH 7.4) at 25ꢂC. Reactions were quenched
with 1 N HCl, and the ratio between substrate (inosine) and product
(hypoxanthine) was quantified by reverse-phase HPLC.
c kiso and khyd were calculated as indicated in Supplemental Data.
d From Kline and Schramm (1992), at 30ꢂC.
e N3-Isoinosine was not detectable by 1H or 13C NMR during the time
course; the limit for detection was estimated to be ꢁ25 mM (0.5% of
the initial [inosine]).
PNPs also showed formation of N3-isoinosine [5] but not ribose
(Figures 5-I and 5-II, 5E, and 5F). N3-Isoinosine is apparent after
ꢁ3 3 106 catalytic turnovers in the phosphorolysis reactions
catalyzed by both H257G and F200G. After the entire inosine
sample had passed through the catalytic site approximately
4000 times (ꢁ24 hr), the N3-isoinosine peak was integrated to
be ꢁ5% and ꢁ10% for H257G and F200G, respectively, relative
to the initial [10-13C]inosine peak (Figure 5); thus, the probability
for N3 capture of the ribocation is 1:80,000 for H257G and
1:40,000 for F200G. In contrast, the phosphorolysis reaction of
native human PNP showed no detectable N3-isoinosine or
ribose formation after more than 107 transits through the cata-
lytic site.
Single-Turnover Hydrolysis
Rates of single-turnover inosine hydrolysis catalyzed by native
and catalytic-site glycine mutants of human PNP were compared
with the previously reported values for bovine PNP (Table 1). The
single-turnover hydrolysis rates for native human PNP were
found to be ꢁ3-fold slower than those for the bovine enzyme
(Table 1, Figure 6A). The introduction of solvent leaks into the
active site of human PNP through the four engineered glycine
mutants caused increased rates of inosine hydrolysis compared
to native PNP (Table 1 and Figure 6A). The rate of hydrolysis for
Y88G increased 2.3-fold, for F159G increased by 4-fold, for
H257G decreased by 1.6-fold, and for F200G increased by
4.8-fold, relative to native PNP (Table 1 and Figure 6A).
1
Figure 4. 13C and H NMR Spectra of [10-13C]Inosine with Human
PNP in the Absence of Phosphate
(Panel I) C NMR spectra of [10-13C]inosine with native human PNP after (A)
13
30 min and (B) 24 hr.
1
(Panel II) H NMR spectra of [10-13C]inosine with native human PNP after (A)
30 min, (B) 3 hr, (C) 6 hr, (D) 12 hr, and (E) 24 hr.
(Panel III) 1H NMR spectra of [10-13C]inosine with F200G after (A) 40 min and (B)
13C NMR Detection of Hydrolysis under High Enzyme
Concentrations
1
24 hr. All 13C and H spectra were acquired with 5.3 mM [10-13C]inosine and
6 mM PNP in the absence of phosphate at 25ꢂC in 10% D2O. [1] Ino, inosine;
To analyze the PNP reaction for slow hydrolysis, 0.9 mM
[10-13C]inosine, 0.2 mM (monomers) of PNPs, and increased
[5] N3-Ino, N3-inosine.
974 Chemistry & Biology 16, 971–979, September 25, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
Ribocation Capture in Human PNP
Figure 5. 13C NMR (I) and 1H NMR (II) Spectra of
[10-13C]Inosine with Human PNP in the Presence of
Phosphate
[10-13C]Inosine (5.3 mM) was incubated (A) alone or with (B)
6 mM native human PNP, (C) 14 mM Y88G, (D) 33 mM F159G,
(E) 151 mM H257G, or (F) 264 mM F200G. All spectra were
acquired in the presence of 50 mM KH2PO4, pH 7.4, at 25ꢂC
in 10% D2O and after ꢁ2
3
107 enzymatic turnovers
(20–24 h). [1] Ino, inosine; [2] Hx, hypoxanthine; [4] R-1-P,
a-D-ribose 1-phosphate; [5] N3-Ino, N3-isoinosine.
example for F159G). Attempts to perform similar
solvolysis experiments with aqueous mixtures of
methanol (up to 20% v/v) failed to yield any detect-
able methyl riboside by 13C or 1H NMR, even after
prolonged incubations (>48 hr).
DISCUSSION
Inosine Hydrolysis by Human PNP
Without phosphate, solvent H2O is expected to fill
the relatively large phosphate binding site of PNP.
From its position in the phosphate site, water acts
as an inefficient nucleophile to capture the riboca-
tion. In contrast, bound phosphate is known to be
highly polarized and activated at the catalytic site
(Deng, et al., 2004). Phosphate also participates
in ribocation formation and geometric stabilization
by forming an ion pair with the developing riboca-
tion and H-bonds to the ribosyl 20- and 30-hydroxyls
(Figure 2). Our results suggest that when these
interactions are missing, the ribosyl group might
not be highly constrained in the catalytic site, and
once it forms, it can rebound to form inosine
(Figure 1, path a), react with water (path c), or react
with N3 of hypoxanthine to form N3-isoinosine
(path d; see below). Interestingly, Richard and
coworkers have also characterized the partitioning
acquisition times were used. These conditions enable 13C-NMR of an enzyme-generated oxacarbenium ion among reactant
detection of ribose resulting from the hydrolysis of a single equiv- reformation, capture by various nucleophiles and capture by
alent of inosine, even if subsequent turnovers are hindered by solvent water (Richard, et al., 1996).
rate-limiting hypoxanthine release, as previously found for
Human PNP hydrolyzes inosine 2.7-fold more slowly than the
bovine PNP (Kline and Schramm, 1995). The 13C NMR spectra bovine enzyme (Kline and Schramm, 1992, 1995). The hydrolytic
with [10-13C]inosine under these conditions showed the appear- reaction rate of 7.5 3 10ꢀ4 sꢀ1 for native HsPNP is approximately
ance of D-ribose [3] (b-D-ribopyranose, d 94.22) (Figure 6B). 5 3 10ꢀ6 of the on-enzyme chemical turnover rate of the phos-
After 20 hr, H257G had converted all of the 0.9 mM inosine to phorolytic reaction (154 sꢀ1) (Saen-Oon, et al., 2008a). Bound
D-ribose [3] (b- and a-D-ribopyranose) (Figure 6B, spectrum phosphate therefore contributes a factor of approximately 105
E). Although inosine hydrolysis by H257G is 1.6-fold slower to TS formation.
than native PNP, it formed hypoxanthine and ribose without
any detectable isomerization to N3-inosine. Native human, Ribocation Reactivity
bovine, and the other three catalytic-site glycine mutants of Transition state lifetimes of nonenzymatic chemical reactions
PNP showed incomplete hydrolysis of [10-13C]inosine [1] and have been shown to be ꢁ10ꢀ13 s, as determined with the use
accumulation of D-ribose [3] (Figure 6B). N3-Isoinosine [5] was of femtosecond time-resolved spectroscopy (Baumert, et al.,
also readily detected with native (human or bovine) PNPs and 2001). Postulates that enzymes preferentially stabilize their
to a lesser extent with F200G (Figure 6B). 13C NMR spectra of transition states suggest that their lifetimes may be substan-
similar experiments for Y88G, F159G and F200G recorded after tially longer. Kinetic isotope effect studies indicate that the TS
48 hr showed significant accumulations of ribose and N3-isoino- of human PNP is a fully dis0sociated ribocation with complete
˚
sine at approximately equal concentrations (Figure S2, as an leaving-group departure (C1 ꢀN9 distance = 3 A) and a similar
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Chemistry & Biology
Ribocation Capture in Human PNP
Figure 6. Hydrolysis Reactions under High Enzyme
Concentrations
(A) Single-turnover hydrolysis reaction kinetics, plotting
the concentration of hypoxanthine (isolated on a C18
analytical column by reverse-phase HPLC) formed during
the hydrolysis reaction of native (d), Y88G (d), F159G (d),
H257G (d), and F200G human PNP (d) as a function of
mixing time prior to acid quenching. Data were fit to Equa-
tion (1).
(B) 13C NMR spectra of the PNP hydrolysis reactions.
[10-13C]inosine (900 mM) with (A) native bovine PNP
(BtPNP), (B) native human PNP (HsPNP), (C) Y88G, (D)
F159G, (E) H257G, and (F) F200G. All spectra were
acquired after 20 hr at 25ꢂC with 200 mM PNP in 10%
D2O. [1] Ino, inosine; [3], D-ribose; [5] N3-Ino, N3-
isoinosine.
˚
TS analogs (Figure 2) reveal a > 3 A separation
between the bound phosphate and C-10,
a distance that is expected to permit the diffu-
sional approach of water at assay temperatures.
Based on the X-ray structures, phosphate is
poised more closely to the ribocation than struc-
turally observed catalytic site waters. If the ribo-
cation is an intermediate and has a lifetime
permitting the diffusion of solvent or catalytic
site waters to the ribocation, then some ribose
formation would be observed. The lack of
hydrolysis is consistent with a ribocation lifetime
that is too short to allow diffusion of a water
molecule to within reactive distance of the ribo-
cation C-10.
Times for water diffusion are a function of
distance and environment. The shortest trajec-
tory from solvent water to C10 of the ribocation
˚
is approximately 10 A by way of a wide channel
located at the interface of the two adjacent
subunits, surrounded by H257, F1590, F200,
distance to the phosphate nucleophile (i.e., an SN1 mechanism) and Y88. Solvent water must diffuse around the ribosyl ring,
(Lewandowicz and Schramm, 2004; Schramm, 2005). The TS a path that is partially obstructed by phosphate and enzyme resi-
lifetime for human PNP has been estimated by computational dues. An alternative consideration is water bound within the
transition path sampling to be ꢁ10 fs (10ꢀ14 s) with reaction catalytic site, as is presumed to exist in the absence of phos-
coordinate motion occurring over ꢁ70 fs (Saen-Oon, et al., phate. X-ray structures of phosphate-bound PNPs reveal the
0
˚
2008b). On-enzyme chemistry occurs over a much longer time- existence of a structural water within 4.4 A of C-1 (Figure 2B).
scale (154 sꢀ1, once every 6 3 10ꢀ3 s), reflecting the necessity Diffusion of approximately 3.0 A would permit formation of
˚
˚
for multiple geometric alignments between the protein and ribose. The upper rate of water diffusion is approximately 1 A
reactants to permit formation of the TS (Saen-Oon, et al., psꢀ1 (based on the water diffusion coefficient of 3.01 3 10ꢀ5
2008a, 2008b). Because the lifetime of the ribocation is cm2$sꢀ1 at 25ꢂC [Wang, 1951]), which sets lower limits of 10
between 10ꢀ14 (the computed TS lifetime) and 6 3 10ꢀ3 s, and 3 ps, respectively, for the time of diffusion of solvent water
we designed an experimental test to determine if the ribocation and the catalytic-site water. The lower limit on the rate of diffu-
is stabilized (near 6 3 10ꢀ3 s) or has a lifetime near the time- sion, however, is not defined for the specific environment in
scale of a single bond vibration lifetime (near 10ꢀ14 s). The life- the catalytic site of PNP. If we assume it is slowed by a factor
time of the ribocation can be estimated by catalytic-site muta- of five, we can, with this assumption, set upper limits of 50 and
tions that are designed to permit water access to the reactive 15 ps, respectively, for these water molecules. The lifetimes of
ribocation.
carbohydrate oxacarbenium ions in water are estimated to be
Undetectable inosine hydrolysis in the presence of phosphate ꢁ1 ps (Amyes and Jencks, 1989). Based on our upper estimates
establishes steric or temporal exclusion for water capture of of diffusion time, we can propose that the ribocation formed at
the ribocation. Transition state analysis (Lewandowicz and the TS of PNP has a lifetime less than the 15 to 50 ps required
Schramm, 2004) and X-ray structures of PNP complexed with for diffusional capture by catalytic-site or solvent water.
976 Chemistry & Biology 16, 971–979, September 25, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
Ribocation Capture in Human PNP
Formation of N3-Isoinosine
branch point in the reaction mechanism (Figure 1, path c). The
A control for the ribocation capture experiment is the rate of in- hydrolytic rate is a measure of the water access and reactivity
osine solvolysis in the absence of phosphate. Removal of phos- as well as the lifetime of the highly reactive ribocation. The
phate permits solvent access to the catalytic site and the mutants are reduced in kcat by up to 44-fold relative to the native
unequivocal formation of ribose. Thus, phosphate-free PNP enzyme. All enzymes were capable of inosine hydrolysis with
forms the ribocation, and without phosphate, most is captured rates similar to or faster than native PNP.
by water to give ribose. In addition to the hydrolytic reaction,
The formation of N3-isoinosine in the absence of phosphate is
a novel product, N3-isoinosine, was also formed. The mecha- consistent with the formation of a ribocation TS that reacts with
nism proposed for N3-isoinosine formation involves ribocation hypoxanthine at N9 (to form inosine) or N3 (yielding N3-isoino-
formation in a catalytic site where mutations weaken the sine) or with water (yielding ribose). Unlike the native enzyme,
geometric alignment of the purine base and/or the ribocation isomerization to N3-isoinosine was not observed with any of
(Figure 2). Without tight geometric constraints on the ribocation the mutants under the conditions of the hydrolysis experiments.
and displaced base, nucleophilic rebound can occur to both In the presence of phosphate, the PNP species with relatively
N9 (the normal reaction) and, by misalignment, to N3, causing high kcat/Km values (i.e., native, Y88G and F159G; Table S1) effi-
isomerization. Phosphate is directly involved in this alignment ciently transfer the ribocation to phosphate without any detect-
by its H-bond contacts with the 20- and 30-hydroxyl groups of able side reactions. Less efficient PNP mutants (i.e., H257G
ribose (Figure 2). In the presence of phosphate, the alignment and F200G) occasionally misdirect the electrophile, resulting in
of N9-hypoxanthine with the ribocation fails less than once in formation of N3-isoinosine. Reaction with N3 is rare—only 1 of
a million transfers through the catalytic site, as evidenced by 40,000 (F200G) or 80,000 (H257G) catalytic cycles generates
the failure to detect any N3-isomer in multiple-turnover NMR N3-inosine, based on its rate of formation. H257 anchors the
studies. In the absence of phosphate, the purine leaving-group 50-hydroxyl group of the ribosyl moiety, and its removal provides
interactions assist in ribocation formation, but the poor reactivity the ribosyl cation sufficient positional flexibility to permit slow
of the disordered waters in the phosphate-binding site allows the ribocation capture by N3. The crystal structure of human PNP
ribocation to wobble on its nucleophilic rebound.
complexed with a TS-analog inhibitor (Immucillin-H) and phos-
The slow formation of N3-isoinosine in the absence of phos- phate indicates that the phenyl-group side chain of residue
phate is consistent with the full formation of the ribocation. F200 is involved in a herringbone-type interaction with the purine
With water molecules in the phosphate site, ribocation capture base (Figure 2) (de Azevedo et al., 2003; Ghanem et al., 2009). In
occurs with N9 (to reform inosine), N3 to form isoinosine, and F200G, the positional stability of the purine base is compro-
solvent to form ribose. In the presence of phosphate, an alterna- mised. In this mutant, the ribocation contacts are unchanged
tive mechanism for isomerization involving N3 attack upon the from native PNP, but weakened interactions to hypoxanthine
ribose 1-phosphate product cannot be excluded, but more permit both N9 and N3 to react with the ribocation.
complex mechanisms depart from known features of the physi-
SIGNIFICANCE
ological PNP mechanism.
N3-Isoinosine is a byproduct in the chemical synthesis of ino-
sine (Lehikoinen et al., 1989; Ryu et al., 2004; Wolfenden et al.,
1966) and is an alternative substrate for PNPs (Bzowska et al.,
1996; Lehikoinen et al., 1989), but it has never been reported
as a product in any enzymatic reactions. Nucleoside deoxyribo-
syltransferases have been demonstrated to form 20-deoxynu-
cleosides with N3- and N7-fused purine derivatives (Huang
et al., 1983; Steenkamp, 1991); however, unlike with PNP, these
isomers are likely formed via the covalent deoxyribosylated
enzyme intermediate that is known to form during its function
with natural substrates (Smar et al., 1991).
Native human PNP and catalytic-site glycine mutants slowly
hydrolyze inosine when phosphate is absent but not in the
presence of phosphate. These mutants were designed to
permit solvent leaks; however, the lifetime of the ribocation
is too short to permit solvent capture of the ribocation in
competition with phosphate. In the absence of phosphate,
human PNP catalyzes the capture of the ribocation by N3
of the purine group, resulting in isomerization of the sub-
strate. The TS of human PNP involves a fully dissociated
ribo-oxacarbenium ion (i.e., SN1 character). The chemical
properties of a ribocation are consistent with the isomeriza-
tion at N3, in which hypoxanthine departure is required to
permit N9 / N3 isomerization. The chemical reactivity of
PNP and its catalytic site mutants provide experimental
evidence for a ribocation lifetime too short to permit efficient
solvent capture of the ribocation. Amino acids that stabilize
both the ribocation (e.g., H257) and hypoxanthine (e.g., F200)
are required for fidelity of product formation. Atomic free-
dom of motion around the TS results in the formation of
N3-isoinosine during phosphorolysis by these mutants.
The lifetime of PNP intermediates can be compared with
a nonenzymatic isomerization reaction for cyclopropane stereo-
mutation (Hrovat et al., 1997). The mechanism of isomerization
proceeds via C–C bond homolysis to generate a trimethylene bir-
adical, which rotates about the linearized bonds before closing.
Their calculations estimated a biradical lifetime of 140 fs, within
the range of the 10–50,000 fs lifetime for the PNP-generated ri-
bocation estimated in our computational (Saen-Oon et al.,
2008b) and solvolysis experiments, respectively.
Inosine Hydrolysis and Isomerization by Catalytic-Site
Mutants of Human PNP
EXPERIMENTAL PROCEDURES
Four glycine mutants of the residues that are proposed to shield
the catalytic site from solvent were prepared to create a water
leak into the catalytic site and thereby introduce a hydrolytic
Steady-State Enzyme Assays
Activity assays for native and mutant PNPs with inosine as a substrate were
carried out by monitoring the conversion of hypoxanthine to uric acid
Chemistry & Biology 16, 971–979, September 25, 2009 ª2009 Elsevier Ltd All rights reserved 977

Chemistry & Biology
Ribocation Capture in Human PNP
(3293 = 12.9 mMꢀ1 cmꢀ1) (Kim et al., 1968) in a coupled assay containing
60 milliunits xanthine oxidase and variable concentrations of inosine in
50 mM KH2PO4 (pH 7.4) at 25ꢂC (Lewandowicz et al., 2003). Substrate
concentration (inosine or [10-13C]inosine) was determined spectrophotometri-
cally using the published millimolar extinction coefficient of 12.3 at 248.5 nm,
pH 7 (Dawson et al., 1986).
buffer (100 mM KH2PO4, 8 mM Bu4NHSO4 [pH 6.0]) at 1 ml minꢀ1, with moni-
toring by a Waters 996 Photodiode Array Detector. Under these conditions,
hypoxanthine, inosine, and N3-isoinosine eluted with retention times of 3.2,
8.07, and 8.8 min, respectively (Figure S1). The N3-isomer peak was collected
and reduced to 400 ml in a Speedvac concentrator, and this sample was de-
salted by application to a second C18 column (Waters Deltapak 15 mm,
300 A, 300 3 3.9 mm), equilibrated with water at 1.5 ml minꢀ1. N3-Isoinosine
˚
[10-13C]Inosine
was collected, concentrated to dryness in a Speedvac concentrator, redis-
solved in water, and analyzed by ESI-MS and by UV/V is spectroscopy at
pH 1, 7, and 11.
[10-13C]Adenosine (Omicron Biochemicals, Inc.) was converted to [10-13C]
inosine by treatment with calf spleen adenosine deaminase (Sigma) (Pfrogner,
1967a, 1967b), with spectrophotometric monitoring at 267 nm (D3267
=
6.5 mMꢀ1 cmꢀ1) (Schramm and Baker, 1985) at pH 7. [10-13C]Inosine was puri-
fied by reverse-phase HPLC (5% methanol in 50 mM triethylammonium
acetate [pH 5.0] at 1 ml minꢀ1) using an analytical C18 column (Waters Delta-
SUPPLEMENTAL DATA
Supplemental Data include three tables, two figures, and Supplemental Exper-
imental Procedures and can be found with this article online at http://www.cell.
com/chemistry-biology/supplemental/S1074-5521(09)00251-8.
˚
pak, 15 mm, 300 A, 300 3 3.9 mm).
Steady-State NMR Detection for Hydrolysis, Phosphorolysis,
and Isomerization
ACKNOWLEDGMENTS
The 13C and 1H NMR spectra of reaction mixtures with PNP and labeled ino-
sine were obtained by mixing 6 mM native human PNP with 5.3 mM [10-13C]ino-
sine in 20 mM Tris-HCl (pH 7.4) and 10% D2O, in the presence or the absence
of 50 mM KH2PO4 (pH 7.4) for ꢁ2 3 107 enzymatic turnovers (ꢁ20 to 24 hr).
Native human PNP has a chemical turnover of 154 sꢀ1 and steady-state rate
of 44 sꢀ1, limited by the rate of hypoxanthine release (Ghanem et al., 2008a).
During 24 hr, the entire inosine sample passes through the catalytic site
approximately 15,000 times (according to chemical turnovers) in this freely
reversible reaction. Thus, a hydrolytic reaction rate of once in every 300,000
We acknowledge the US National Institutes of Health grants GM41916 and
GM068036 for funding.
Received: April 30, 2009
Revised: June 26, 2009
Accepted: July 23, 2009
Published: September 24, 2009
phosphorolytic reactions (5 3 10ꢀ4 ꢀ1) would cause 5% of the inosine to
s
REFERENCES
accumulate as ribose and would be readily detected by NMR. For mutant
PNPs, enzyme was added at concentrations that yield the same steady-state
Amyes, T.L., and Jencks, W.P. (1989). Lifetimes of oxocarbenium ions in
aqueous solution from common ion inhibition of the solvolysis of. alpha.-azido
ethers by added azide ion. J. Am. Chem. Soc. 111, 7888–7900.
phosphorolytic rate as native PNP (e.g., [H257G] = [Native PNP] 3 kcat,native
O
kcat,H257G = 6 mM 3 44 sꢀ1 O 1.75 sꢀ1 = 150 mM). Standard references included
free [10-13C]inosine and [1-13C]ribose obtained through the irreversible arsen-
olysis of [10-13C]inosine in the presence of PNP and 50 mM Na2HAsO4 (pH 7.4).
All spectra were acquired using a Bruker 300 MHz spectrometer with 5 mm BBI
probe at 298 K. 1H-decoupled 13C NMR spectra were collected with 256 scans
of 64 K points (ꢁ15 min/spectrum), a recycle delay of 2 s and a carbon sweep
width of 315 ppm with the carrier set to 126 ppm. 1H NMR spectra were
collected with 64 scans of 16 K points, a recycle delay of 1.3 s and a proton
sweep width of 14 ppm with the carrier set to 4.7 ppm. Spectra were pro-
cessed with an exponential window function of 1 Hz. Spectra were assigned
as described previously (Dorman and Roberts, 1970; Teijeira et al., 1997;
Tindall et al., 1972).
Baumert, T., Frohnmeyer, T., Kiefer, B., Niklaus, P., Strehle, M., Gerber, G.,
and Zewail, A.H. (2001). Femtosecond transition state dynamics of cis-stil-
bene. Appl. Phys. B 72, 105–108.
BioCryst (2009). BioCryst research pipeline (http://www.biocryst.com/
pipeline.htm). BioCryst Pharmaceuticals, Inc.
Bzowska, A., Kulikowska, E., Poopeiko, N.E., and Shugar, D. (1996). Kinetics
of phosphorolysis of 3-(beta-D-ribofuranosyl)adenine and 3-(beta-D-ribofura-
nosyl)hypoxanthine, non-conventional substrates of purine-nucleoside phos-
phorylase. Eur. J. Biochem. 239, 229–234.
Chenon, M.T., Pugmire, R.J., Grant, D.M., Panzica, R.P., and Townsend, L.B.
(1975a). Carbon-13 magnetic resonance. XXV. A basic set of parameters for
the investigation of tautomerism im purines. Established from carbon-13
magnetic resonance studies using certain purines and pyrrolo[2,3-d]pyrimi-
dines. J. Am. Chem. Soc. 97, 4627–4636.
13C NMR Detection for Hydrolysis under High Enzyme
Concentrations
13C NMR spectra of reaction mixtures with high molar ratios of PNP to inosine
were obtained by mixing 200 mM (monomers) of PNP with 900 mM of [10-13C]
inosine in 20 mM Tris-HCl (pH 7.4) and 10% D2O, for ꢁ20 to 24 hr. 1H-de-
coupled 13C NMR spectra were collected with 1024 scans of 64 K points
(ꢁ58 min), a recycle delay of 2 s and a carbon sweep width of 315 ppm with
the carrier set to 126 ppm using a Bruker 300 MHz spectrometer with 5 mm
Chenon, M.T., Pugmire, R.J., Grant, D.M., Panzica, R.P., and Townsend, L.B.
(1975b). Carbon-13 magnetic resonance. XXVI. A quantitative determination of
the tautomeric populations of certain purines. J. Am. Chem. Soc. 97, 4636–
4642.
BBI probe at 298 K. See Supplemental Data for calculation of kiso
.
Dawson, R.M.C., Elliott, D., Elliott, W.H., and Jones, K.M. (1986). Spectral data
and pKa values for purines, pyrimidines, nucleosides, and nucleotides. In Data
for Biochemical Research, Third Edition, R.M.C. Dawson, ed. (New York:
Oxford Univ. Press), pp. 103–114.
Single-Turnover Hydrolysis
Equal amounts of inosine and enzyme (80 mM final) were incubated at 25ꢂC for
30 s and up to 72 hr, followed by quenching with HCl (1 N final) and removal of
the denatured protein by centrifugation. Hypoxanthine (retention time 4.3 min)
and inosine (retention time 7.3 min) were quantitated by UV detection at
249 nm using reverse-phase HPLC on a Waters Deltapak C18 column
de Azevedo, W.F., Jr., Canduri, F., dos Santos, D.M., Silva, R.G., de Oliveira,
J.S., de Carvalho, L.P., Basso, L.A., Mendes, M.A., Palma, M.S., et al. (2003).
Crystal structure of human purine nucleoside phosphorylase at 2.3 A resolu-
tion. Biochem. Biophys. Res. Commun. 308, 545–552.
˚
(15 mm, 300 A, 300 3 3.9 mm), equilibrated with 5% methanol in 50 mM trie-
thylammonium acetate (pH 5.0) at 1 ml minꢀ1. See Supplemental Data for
Deng, H., Lewandowicz, A., Schramm, V.L., and Callender, R. (2004). Acti-
vating the phosphate nucleophile at the catalytic site of purine nucleoside
phosphorylase: a vibrational spectroscopic study. J. Am. Chem. Soc. 126,
9516–9517.
calculation of khyd
.
Characterization of N3-Isoinosine
N3-Isoinosine was isolated from incubations of inosine with human PNP in the
Dorman, D.E., and Roberts, J.D. (1970). Nuclear magnetic resonance spec-
troscopy: 13C spectra of some common nucleotides. Proc. Natl. Acad. Sci.
USA 65, 19–26.
absence of phosphate by reverse-phase HPLC using an analytical C18 column
˚
(Phenomenex Luna 5 mm, 100 A, 150 3 4.6 mm), equilibrated with an ion-pair
978 Chemistry & Biology 16, 971–979, September 25, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
Ribocation Capture in Human PNP
Ebrahimi, M., Rossi, P., Rogers, C., and Harbison, G.S. (2001). Dependence of
13C NMR chemical shifts on conformations of rna nucleosides and nucleo-
tides. J. Magn. Reson. 150, 1–9.
Nunez, S., Wing, C., Antoniou, D., Schramm, V.L., and Schwartz, S.D. (2006).
Insight into catalytically relevant correlated motions in human purine nucleo-
side phosphorylase. J. Phys. Chem. A 110, 463–472.
Pfrogner, N. (1967a). Adenosine deaminase from calf spleen. I. Purificiation.
Fedorov, A., Shi, W., Kicska, G., Fedorov, E., Tyler, P.C., Furneaux, R.H., Han-
son, J.C., Gainsford, G.J., Larese, J.Z., et al. (2001). Transition state structure
of purine nucleoside phosphorylase and principles of atomic motion in enzy-
matic catalysis. Biochemistry 40, 853–860.
Arch. Biochem. Biophys. 119, 141–146.
Pfrogner, N. (1967b). Adenosine deaminase from calf spleen. II. Chemical and
enzymological properties. Arch. Biochem. Biophys. 119, 147–154.
Ghanem, M., Li, L., Wing, C., and Schramm, V.L. (2008a). Altered thermody-
namics from remote mutations altering human toward bovine purine nucleo-
side phosphorylase. Biochemistry 47, 2559–2564.
Richard, J.P., Huber, R.E., Heo, C., Amyes, T.L., and Lin, S. (1996). Structure-
reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 4.
Mechanism for reaction of nucleophiles with the galactosyl-enzyme intermedi-
ates of E461G and E461Q beta-galactosidases. Biochemistry 35, 12387–
12401.
Ghanem, M., Saen-oon, S., Zhadin, N., Wing, C., Cahill, S.M., Schwartz, S.D.,
Callender, R., and Schramm, V.L. (2008b). Tryptophan-free human PNP
reveals catalytic site interactions. Biochemistry 47, 3202–3215.
Rinaldo-Matthis, A., Murkin, A.S., Ramagopal, U.A., Clinch, K., Mee, S.P.,
Evans, G.B., Tyler, P.C., Furneaux, R.H., Almo, S.C., et al. (2008). L-Enantio-
mers of transition state analogue inhibitors bound to human purine nucleoside
phosphorylase. J. Am. Chem. Soc. 130, 842–844.
Ghanem, M., Zhadin, N., Callender, R., and Schramm, V.L. (2009). Loop-tryp-
tophan human PNP reveals submillisecond protein dynamics. Biochemistry
48, 3568–3668.
Ryu, K.-S., Kim, C., Park, C., and Choi, B.-S. (2004). NMR analysis of enzyme-
catalyzed and free-equilibrium mutarotation kinetics of monosaccharides.
J. Am. Chem. Soc. 126, 9180–9181.
Hrovat, D.A., Fang, S., Borden, W.T., and Carpenter, B.K. (1997). Investigation
of cyclopropane stereomutation by quasiclassical trajectories on an analytical
potential energy surface. J. Am. Chem. Soc. 119, 5253–5254.
Saen-Oon, S., Ghanem, M., Schramm, V.L., and Schwartz, S.D. (2008a).
Remote mutations and active site dynamics correlate with catalytic properties
of purine nucleoside phosphorylase. Biophys. J. 94, 4078–4088.
Huang, M.C., Montgomery, J.A., Thorpe, M.C., Stewart, E.L., Secrist, J.A., 3rd,
and Blakley, R.L. (1983). Formation of 3-(20-deoxyribofuranosyl) and 9-(20-de-
oxyribofuranosyl) nucleosides of 8-substituted purines by nucleoside deoxyri-
bosyltransferase. Arch. Biochem. Biophys. 222, 133–144.
Saen-Oon, S., Quaytman-Machleder, S., Schramm, V.L., and Schwartz, S.D.
(2008b). Atomic detail of chemical transformation at the transition state of an
enzymatic reaction. Proc. Natl. Acad. Sci. USA 105, 16543–16548.
Kenneth, N., Drew, J.Z., Bondo, G., Bose, B., and Serianni, A.S. (1998).
13C-labeled aldopentoses: detection and quantitation of cyclic and acyclic
forms by heteronuclear 1D and 2D NMR spectroscopy. Carbohydr. Res.
307, 199–209.
Schramm, V.L. (2005). Enzymatic transition states: thermodynamics,
dynamics and analogue design. Arch. Biochem. Biophys. 433, 13–26.
Schramm, V.L., and Baker, D.C. (1985). Spontaneous epimerization of (S)-de-
oxycoformycin and interaction of (R)-deoxycoformycin, (S)-deoxycoformycin,
and 8-ketodeoxycoformycin with adenosine deaminase. Biochemistry 24,
641–646.
Kim, B.K., Cha, S., and Parks, R.E., Jr. (1968). Purine nucleoside phosphory-
lase from human erythroyctes. II. Kinetic analysis and substrate-binding
studies. J. Biol. Chem. 243, 1771–1776.
Kline, P.C., and Schramm, V.L. (1992). Purine nucleoside phosphorylase. Ino-
sine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-
sites reactivity. Biochemistry 31, 5964–5973.
Shi, W., Ting, L.M., Kicska, G.A., Lewandowicz, A., Tyler, P.C., Evans, G.B.,
Furneaux, R.H., Kim, K., Almo, S.C., et al. (2004). Plasmodium falciparum
purine nucleoside phosphorylase: crystal structures, Immucillin inhibitors,
and dual catalytic function. J. Biol. Chem. 279, 18103–18106.
Kline, P.C., and Schramm, V.L. (1993). Purine nucleoside phosphorylase.
Catalytic mechanism and transition-state analysis of the arsenolysis reaction.
Biochemistry 32, 13212–13219.
Smar, M., Short, S.A., and Wolfenden, R. (1991). Lyase activity of nucleoside
2-deoxyribosyltransferase: transient generation of ribal and its use in the
synthesis of 20-deoxynucleosides. Biochemistry 30, 7908–7912.
Kline, P.C., and Schramm, V.L. (1995). Pre-steady-state transition-state anal-
ysis of the hydrolytic reaction catalyzed by purine nucleoside phosphorylase.
Biochemistry 34, 1153–1162.
Steenkamp, D.J. (1991). The purine-2-deoxyribonucleosidase from Crithidia
luciliae. Purification and trans-N-deoxyribosylase activity. Eur. J. Biochem.
197, 431–439.
Koellner, G., Luic, M., Shugar, D., Saenger, W., and Bzowska, A. (1997).
Crystal structure of calf spleen purine nucleoside phosphorylase in a complex
with hypoxanthine at 2.15 A resolution. J. Mol. Biol. 265, 202–216.
Stoeckler, J.D., Agarwal, R.P., Agarwal, K.C., and Parks, R.E., Jr. (1978).
Purine nucleoside phosphorylase from human erythrocytes. Methods Enzy-
mol. 51, 530–538.
Lehikoinen, P.K., Sinnott, M.L., and Krenitsky, T.A. (1989). Investigation of
a-deuterium kinetic isotope effects on the purine nucleoside phosphorylase
reaction by the equilibrium-perturbation technique. Biochem. J. 257, 355–359.
Stoeckler, J.D., Cambor, C., and Parks, R.E., Jr. (1980). Human erythrocytic
purine nucleoside phosphorylase: reaction with sugar-modified nucleoside
substrates. Biochemistry 19, 102–107.
Lewandowicz, A., and Schramm, V.L. (2004). Transition state analysis for
human and Plasmodium falciparum purine nucleoside phosphorylases.
Biochemistry 43, 1458–1468.
Teijeira, M., Santana, L., and Uriarte, E. (1997). Assignment of the 13C NMR
spectra of some adenine, hypoxanthine and guanine carbonucleosides.
Magn. Reson. Chem. 35, 806–807.
Lewandowicz, A., Shi, W., Evans, G.B., Tyler, P.C., Furneaux, R.H., Basso,
L.A., Santos, D.S., Almo, S.C., and Schramm, V.L. (2003). Over-the-barrier
transition state analogues and crystal structure with Mycobacterium tubercu-
losis purine nucleoside phosphorylase. Biochemistry 42, 6057–6066.
Tindall, C.G., Jr., Robins, R.K., Tolman, R.L., and Hutzenlaub, W. (1972).
Directed glycosylation of 8-bromoadenine. Synthesis and reactions of
8-substituted 3-glycosyladenine derivatives. J. Org. Chem. 37, 3985–3989.
Montgomery, J.A., and Thomas, H.J. (1969). Ribosyl derivatives of hypoxan-
Wang, J.H. (1951). Self-diffusion and structure of liquid water. II. Measurement
of self-diffusion of liquid water with O18 as tracer. J. Am. Chem. Soc. 73, 4181–
4183.
thine. J. Org. Chem. 34, 2646–2650.
Murkin, A.S., Birck, M.R., Rinaldo-Matthis, A., Shi, W., Taylor, E.A., Almo, S.C.,
and Schramm, V.L. (2007). Neighboring group participation in the transition
state of human purine nucleoside phosphorylase. Biochemistry 46, 5038–
5049.
Wolfenden, R., Sharpless, T.K., Ragade, I.S., and Leonard, N.J. (1966). Enzy-
matic and chemical deamination of 3-(b-D-ribofuranosyl)adenine. J. Am.
Chem. Soc. 88, 185–186.
Chemistry & Biology 16, 971–979, September 25, 2009 ª2009 Elsevier Ltd All rights reserved 979