Angewandte
Chemie
Table 1: Kinetic parameters for the PAS-catalyzed hydrolysis of phos-
[
a]
phodiesters 1, sulfate monoester 2, and phosphate monoester 3.
À1
À1 À1
[b]
Substrate Km [mm]
kcat [s
]
kcat/Km [m
s
]
(kcat/K )/k
m 2
1
5
1
1
1
2
3
a
b
c
617Æ61
2.2Æ0.3
25Æ2
0.073Æ0.004 119Æ7
6.5ꢀ10
1.3ꢀ10
5
18
0.55Æ0.02
0.19Æ0.01
(2.5Æ0.3)ꢀ10
(7.6Æ0.5)ꢀ10
(4.9Æ0.8)ꢀ10
3
7
Figure 2. Transition states during phosphodiester (associative; left)
and sulfate monoester hydrolysis (dissociative; right).
[
c]
18
13
0.29Æ0.03 14.2Æ0.6
4.3ꢀ10
1.6ꢀ10
[
c]
29.1Æ2.0
0.023Æ0.001 790Æ58
À1
[
a] Reaction conditions: 258C, 0.1m Tris-HCl (pH 8.0), 0.5 mgmL
[
15]
15
bovine serum albumin (BSA), 0.5m NaCl (for 1a–c). [b] For hydrolytic
lyzes phosphate diesters ((k /K )/k = 8 ꢀ 10 ) with lower
cat m 2
reactions, the second-order rate constant k for the uncatalyzed reaction
sulfate and phosphate monoesterase activities (proficiencies
less than 10 ).
2
À1
[11,14]
is taken as k /55m. Values for k /s at 258C, pH 8.0:
1.0ꢀ
11 [20,21]
uncat
uncat
À10
À12
À11
À9
1
0
(1a), 1.1ꢀ10
(1b), 6.2ꢀ10
(2), 2.7ꢀ10 (3). [c] Data for
It is remarkable that PAS can achieve large and com-
parable rate enhancements for two reactions with distinct
features. Both catalyzed reactions are hydrolytic, but the
intrinsic chemistry, which is defined by bond-making and
bond-breaking, involves mechanistic alternatives. In solution,
the hydrolysis of phosphodiesters 1 occurs via a transition
state that is more associative in nature, with little negative
[
14]
sulfate (2) and phosphate (3) monoesters are included for compar-
ison.
(
Supporting Information, Figure S1). A phosphate diester
substrate acts as a competitive inhibitor of the native sulfatase
activity of PAS (Supporting Information, Figures S2 and S3),
indicating that both reactions occur in the same active site.
Furthermore, a mutant, C51S, lacking the wild-type nucleo-
phile (see Scheme 2) has a reduced kcat value for the reactions
[22–24]
charge development on the leaving group.
By contrast,
sulfate and phosphate monoesters proceed through dissocia-
tive transition states, with significant negative charge on the
[
14]
[22,25,26]
with both phosphate diester 1b and sulfate monoester 2 (by
leaving group (Figure 2).
PAS displays greater selec-
4
3
[16]
more than 10 and 10 respectively; Table 2).
tivity for substrate structure than for the transition-state
characteristics of the background reactions: we observed a
3
1
0 -fold variation in k /K for diesters 1a–c, with an
cat
m
apparent preference for diesters with two aromatic rings,
yet the values for diester 1b and sulfate 2 differ by just 100-
fold. Substrate charge appears to have a large influence on
reactivity; the increase in charge on phosphate monoester 3
relative to sulfate 2 is penalized by a 6 ꢀ 10 -fold decrease in
k /K , despite both reactions occurring via a dissociative
cat m
[
a]
Table 2: Kinetic parameters for the C51S mutant of PAS.
À1
À1 À1
Substrate
Km [mm]
kcat [s
]
kcat/Km [m
s
]
À5
1
2
b
3.7Æ0.3
(<5.5Æ0.1)ꢀ10
<15Æ1
[
b]
À3
4
4
0.25Æ0.06
(5.4Æ0.2)ꢀ10
(2.1Æ0.5)ꢀ10
À1
[
a] Reaction conditions: 258C, 0.1m Tris-HCl (pH 8.0), 0.5 mgmL BSA,
0
.5m NaCl (for 1b). [b] Data for sulfate monoester 2 included for
transition state in solution. The nature of the transition states
for PAS-catalyzed reactions remain to be determined.
[
14]
comparison.
[
27,28]
[22,29]
[30]
AP,
many other phosphatases,
and a sulfatase use
similar mechanisms to those in solution, but an example of a
promiscuous enzyme that hydrolyzes phosphate and phos-
phonate monoesters with transition states that are closer to
one another than those of the non-enzymatic reaction was
Catalytic promiscuity is a familiar feature in PAS, which
also hydrolyzes phosphate monoesters, such as 3, but much
[
14]
less efficiently than sulfate monoesters, such as 2.
The
[
31]
catalytic proficiencies (k /K )/k of the enzymatic reactions
recently reported.
cat
m
2
1
3
18
for substrate 3 is 1.6 ꢀ 10 and 4.3 ꢀ 10 for 2 (Table 1).
However, the phosphodiesterase activity (1b) of PAS has an
Efficient hydrolysis of phosphate diesters by PAS may
result from active-site groups that could carry out similar roles
during catalysis of both native and promiscuous reactions.
1
8
exceptionally high catalytic proficiency of 1.3 ꢀ 10 . Promis-
cuous cyclic phosphodiesterase activity has been previously
Specifically, the proposed double displacement mechanism
[
17]
[32]
observed for a sulfatase, but with lower efficiency.
This
for sulfate hydrolysis
involves Lewis acid catalysis by a
value surpasses the rate accelerations observed for the native
reactions of many enzymes, and to our knowledge is greater
than the proficiency of any previously reported promiscuous
calcium ion, a reactive nucleophile, and efficient substrate
binding and general acid catalysis by several cationic active
site groups (Supporting Information, Figure S4). The
decreased activity of the C51S mutant confirms the key role
of the formylglycine (FGly) nucleophile, which is formed by
[
6,11]
enzyme activity.
In comparison, two structurally and
evolutionarily related enzymes that belong to the same
superfamily as PAS catalyze the same three hydrolytic
reactions but show greater discrimination between the
native and promiscuous activities. E. coli alkaline phospha-
tase (AP) is a native phosphate monoesterase that also
[33]
post-translational modification of cysteine 51,
in both
reactions. This nucleophile can be regenerated by hemiacetal
cleavage, enabling multiple turnovers per active site
(Scheme 2 and inset in Figure 1).
The substantial rate accelerations show that PAS is a
highly efficient catalyst for both sulfate monoester and
[
18,19]
hydrolyzes sulfate monoesters and phosphate diesters,
but the proficiencies for the promiscuous reactions are at least
6
1
0 -fold lower than for the native phosphatase activity ((k /
phosphate diester hydrolysis. This efficiency is also reflected
cat
1
7
À17
K )/k = 7 ꢀ 10 ). Xanthomonas axonopodis nucleotide phos-
m
in the tight transition state binding (K = 1.3 ꢀ 10 m and
2
tx
À17
phodiesterase/pyrophosphatase (NPP) preferentially hydro-
4.2 ꢀ 10 m for substrates 1b and 2, respectively). This
Angew. Chem. Int. Ed. 2009, 48, 3692 –3694
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3693