Catechol oxidase activity of Di-Cu2+-substituted aminopeptidase from Streptomyces griseus

Article abstract of DOI:10.1021/ja056034u

Streptomyces griseus aminopeptidase exhibits activities toward the hydrolyses of peptides and bis(p-nitrophenyl)phosphate (40 billion fold) and catechol oxidation reported herein with catalytic efficiency (k_cat/K_m) only about 10 times smaller than that of gypsywort catechol oxidase. The multifunctionality of this enzyme suggests that it is a unique system for further exploration of protein structure and function and a template for design of enzymes of diverse activities. Copyright

Full text of DOI:10.1021/ja056034u

Published on Web 11/04/2005
Catechol Oxidase Activity of Di-Cu2+-Substituted Aminopeptidase from
Streptomyces griseus
Giordano F. Z. da Silva and Li-June Ming*
Department of Chemistry and Institute for Biomolecular Science, UniVersity of South Florida, 4202 East Fowler
AVenue, Tampa, Florida 33620-5250
Received September 1, 2005; E-mail: ming@shell.cas.usf.edu
The predominant theory concerning enzymatic catalysis is that
enzymes have evolved to perform specific chemical transformations
on their respective substrates by stabilizing the transition state
(TSq),1 such as the tetrahedral TSq during peptide hydrolysis by
metallohydrolases. However, several examples of enzyme catalytic
promiscuity,2 that is, the catalyses of more than one type of reactions
by a single enzyme, have been observed, particularly within enzyme
superfamilies which can be mostly attributed to divergent evolution.2
Nevertheless, these results still reflect that if the TSq of an alternative
reaction can be stabilized by an enzyme the reaction may take place
effectively. While many metal-substituted derivatives of metal-
loenzymes are active, some are inert, which nevertheless can provide
structural information regarding the enzyme-substrate complexes
that may not be readily available from active derivatives.3 Metal
substitution has thus been widely used to shed light on the structure
and mechanism of metalloproteins.4 Using apo metalloprotein
molecules as natural ligands and tuning their structures/activities
with the active-site metals are unique approaches toward exploration
Figure 1. (A) Aerobic oxidation of DTC (O, pH 7.0, left scale) and catechol
and discovery of new metalloprotein systems. However, altering
(b, pH 8.0, right scale) by CuCu-SgAP.9 The inset shows competitive
catalytic specificity based on simple metal substitution of metal-
inhibition of DTC oxidation by dinuclear AP-specific bestatin at 0.0, 5.0,
10.0, and 20.0 µM (from bottom), similar to the inhibition toward peptide
loenzymes was rarely reported in the literature.5 Recently, the
hydrolysis. (B) Metal titration to apo-SgAP in 100.0 mM HEPES at pH
7.0 and monitored with Leu-pNA hydrolysis and DTC oxidation, and fitted
to Cd2+-binding pattern previously published.13
dinuclear aminopeptidase from Streptomyces griseus (SgAP) was
found to exhibit a catalytic promiscuity toward phosphoester
hydrolysis with a catalytic proficiency >40 billion under physi-
ological conditions.6 Since phosphoesters are structurally and
mechanistically different from peptides during hydrolysis, this
catalytic promiscuity is quite unusual. Herein we present that di-
Cu2+-substituted SgAP (CuCu-SgAP) exhibits a remarkable oxida-
tive activity, approaching that of native catechol oxidase. The
multifunctional character of SgAP and its metal derivatives makes
this enzyme an exceptional candidate for further exploration of
protein structure and function in combination with molecular
biology techniques.
The purification of SgAP7,8 and preparation of its metal deriva-
tives8 followed published procedures. The oxidation of the proto-
typical catechol oxidase substrate, 3,5-di-tert-butylcatechol (DTC),
by CuCu-SgAP follows Michaelis-Menten kinetics (O, Figure
1A),9 affording kcat ) 1.45 s-1 and Km ) 0.44 mM. The catalytic
efficiency of this catalysis (kcat/Km ) 3295 M-1 s-1) under mild
conditions at 25 °C and pH 7.0 in 50.0 mM HEPES and 5.0 mM
Ca2+ is much better than that of a number of synthetic metal
complexes10 and is only ∼10 times smaller than that of catechol
oxidase from gypsywort (32 mM-1 s-1).11
hydrolytic activity controlled by the relative magnitude of the two
metal-binding constants.8,13 The oxidation reaction reaches full
activity with the addition of 2 equiv of Cu2+ and can be well fitted
as in the hydrolytic catalysis (Figure 1B). The data indicate that
the two completely different reactions, hydrolysis and oxidation,
are carried out by a single enzyme with a dimetal active center.
Both hydrolytic and oxidative activities are found to be com-
petitively inhibited by the dinuclear AP-specific inhibitor bestatin
(inset, Figure 1A) with similar Ki values, 11.0 µM for Leu-pNA
hydrolysis and 8.8 µM for DTC oxidation. This observation
indicates that the inhibitor and the two substrates bind to the active
site in a similar way in these two reactions. SgAP is specific toward
hydrophobic amino acids based on kinetic7 and crystallography14
studies. The hydrophobic pocket for specific recognition in SgAP
may facilitate the binding of DTC to the active site via its tert-
butyl groups. DTC binding can also be promoted via deprotonation
of the OH groups in DTC facilitated by the strong Lewis acidic
Cu2+. The structural similarity between these two substrates can
be clearly shown when they are superimposed to each other (Figure
2). To further demonstrate this point, the rate of catechol oxidation
is measured,9 which gives kcat ) 0.066 s-1, Km ) 0.021 mM, and
kcat/Km ) 3220 M-1 s-1 at pH 8.0 (b, Figure 1A); however, it is
not noticeable at pH 7.0. The decrease in Km compared to that at
To demonstrate that the high oxidative activity observed herein
is indeed attributed to SgAP, activity profiles were obtained in
stoichiometric Cu2+ titration of apo-SgAP (Figure 1B) and ther-
modeactivation.12 The results show that the activities toward the
oxidation of DTC and the hydrolysis of Leu-p-nitroanilide8,13 (Leu-
pNA) are parallel. We have previously shown that metal ions bind
sequentially to the two metal-binding sites of apo-SgAP, with the
pH 7.0 is probably due to the decrease in kcat since Km ) (kcat
k-1)/k1 in Michaelis-Menten kinetics. CuCu-SgAP can also oxidize
a biorelevant catechol, dopamine, with kcat ) 0.097 s-1 and Km
+
)
9
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C O M M U N I C A T I O N S
all-His vs a mixed His/carboxylato environment), the catalytic
promiscuity observed here reflects that the active site does not need
to be restricted to one structural pattern, which thus affords a new
direction for rational design of ligands and proteins for oxidation
catalysis. SgAP and its metal derivatives are the only protein system
to date that shows sharply different multiple catalytic activities.
Thus, this enzyme and its variants prepared in the future can serve
as unique dinuclear systems to provide further insight into the
correlation of structure and mechanism of metal-centered hydrolytic
and oxidation/oxygenation chemistry and may also serve as a “living
fossil” to hint for divergent enzyme evolution.
Figure 2. DTC superimposed onto Leu to reveal their structural similarity
(BioMedCAChe 6.1.10). In Leu, the carboxylate and the amino groups are
for metal binding, and the isobutyl group is for hydrophobic recognition.
Acknowledgment. This work was partially supported by the
Petroleum Research Funds, American Chemical Society (ACS-PRF
#35313-AC3), and the National Institutes of Health (GM064400-
01A2). This work is dedicated to Professor Joan Selverstone
Valentine on the occasion of her 60th birthday.
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(9) (a) Oxidation of the catechol moiety can be monitored directly at 420 nm
(ꢀ ) 1600 M-1 cm-1) for the corresponding o-quinone or at 500 nm (ꢀ )
32 500 M-1 cm-1) in the presence of the o-quinone indicator,9b 3-methyl-
2-benzothiazolinone hydrazone MBTH (Fisher, Pittsburgh, PA), on a
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were calculated and fitted to the Michaelis-Menten equation to give the
rate constants. A typical assay contained 80.0 nM CuCu-SgAP in 50.0
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Figure 3. 4,5-Dichlorocatechol (DCC) titration to 0.1 mM CuCu-SgAP in
50.0 mM HEPES at pH 7.0. The inset is the best fit to a quadratic ligand
binding pattern showing 1:1 DCC:CuCu-SgAP stoichiometry.
0.60 mM and a small catalytic efficiency of 162 M-1 s-1 at pH
7.0, which might be attributed to its different metal-binding and
recognition from Leu (cf. Figure 2).
The oxidation of DTC showed a [H2O2]-dependent increase in
activity. In the presence of 10.0 mM (0.034%) H2O2, the kcat value
of CuCu-SgAP is further increased (2.03 s-1) with only a small
change in Km (0.32 mM), which affords a second-order rate constant
nearly doubled to 6344 M-1 s-1. The proposed mechanism of
catechol oxidase includes an active Cu2+2-µ-η2-η2-peroxo species
that is isoelectronic to Cu+2O2 and Cu3+-bis-µ-oxo, which are all
capable of performing the 2e- oxidation of catechol to yield
o-quinone.15 Herein, H2O2 may facilitate the formation of an
electrophilic Cu2+2-µ-peroxo intermediate in CuCu-SgAP.
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4,5-dichlorocatechol (DCC which is only oxidized slowly at pH
8.0 with kcat ) 0.0045 s-1 and Km ) 0.15 mM), was titrated into
0.1 mM CuCu-SgAP at 25 °C and pH 7.0, and the electronic spectra
were collected (Figure 3). The ligand-to-metal charge transfer band
at 437 nm increases upon addition of DCC and reaches saturation
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fitted to a single-substrate binding mode (Figure 3, inset) to yield
a dissociation constant of 0.13 mM. The result provides direct
evidence and stoichiometry (DCC:active site ) 1:1) for catechol
binding to the dinuclear active site of CuCu-SgAP, yielding ꢀmax
) 1200 M-1 cm-1, that is consistent with observations in DCC or
trichlorocatechol binding to a dinuclear Cu2+ center.16
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can serve as a template for the design of potential catalysts capable
of performing various chemical transformations, such as hydrolyses6
of peptide, phosphodiester, and phosphonatoester and catechol
oxidation, due to its easy purification, high thermal stability,7 and
easy preparation of metal derivatives.8,13 The existence of a “met-
like” di-Cu site in CuCu-SgAP similar to that observed in the crystal
structure of catechol oxidase16 with a bridging OH between the
two Cu2+ centers likely plays a role in the catalysis. Nevertheless,
since the active sites of these two enzymes are quite different (an
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110.
(12) After 7 h treatment at 70 °C, 21 and 23% activity remains, respectively,
toward the hydrolysis of Leu-pNA and oxidation of DTC.
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120-127.
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