A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 12 (2002) 1177–1180
1179
increase of affinity. This increase was indeed drastic for
the tetrapeptides DADD and DDDD, which show affi-
nity of 0.1–0.2 mM against hCA II, being thus very
active CAAs. Furthermore, the Boc-derivatized com-
pounds 15–19 or the N-acetylated dipeptides Ac-DA
and Ac-DD (obtained from the corresponding dipep-
tides and acetic anhydride) exhibited much weaker CA
activatory properties as compared to the corresponding
deprotected compounds (Table 1).
Two main conclusions may be drawn from the above
data. First, this is the first time that activators posses-
sing higher affinity for hCA II than for hCA I/bCA IV
are detected. Indeed, in many previous series of deriva-
tives investigated by us, the range of affinity of a given
Figure 1. hCA II active site. The Zn(II) ion (central pink sphere) and
its three histidine ligands (in green, His 94, His 96, His 119) are shown.
The histidine cluster, comprising residues His 64, His 4, His 3, His 17,
His 15 and His 10, is also evidenced, as this is considered to play a
critical role in binding activators of the type 6–14, reported in the
paper as well as the carboxyterminal part of the anion exchanger AE1.
The figure was generated from the X-ray coordinates reported by this
group (PDB entry 4TST).8
activator for the three isozymes was hCA I>bCA IV
13À15,17
>
>hCA II.
For this small series of oligopeptide
CAAs, the affinity is in the order: hCA II>hCA
I>bCA IV. Second, one of the best activators of this
series, DADD, possesses just the sequence recently evi-
denced to be critical for the binding of hCA II to the
1
2
bicarbonate/chloride anion exchanger AE1. Our data
strongly suggest that in the complex formed by the two
proteins, the carboxyterminal part of AE1 actually has
the function of a natural CAA, facilitating catalysis and
formation of bicarbonate, which is then transported by
the AE1 out of the red cell. The two enzymes form in
this way a metabolon, a weakly associated complex of
sequential metabolic enzymes. We want to stress again
that the CA II activating properties of the Ct part of
AE1 has not been taken into consideration up to now.
catalytic hydrogenation (Scheme 1). The Boc-deriva-
tives 15–19 were obtained from the corresponding
amino acid/oligopeptides and Boc-On, whereas the
acetylated derivatives by reaction of the corresponding
1
3À17,19
21
dipeptides with acetic anhydride.
CA activation
Activation data against three CA isozymes with alanine,
aspartic acid and diverse di-, tri- or tetrapeptides incor-
porating them are shown in Table 1, together with data
of standard CAAs, such as histamine, histidine or car-
nosine. One may see that both aspartic acid as well as
alanine are weaker CAAs as compared to histamine,
As also shown by the work of Reithmeyer’s group,12,21
basic amino acid residues at the aminoterminal part of
CA II are involved in binding of the carboxyterminal
part of AE1, and more precisely of the DADD
sequence. In Figure 1, some of the probable residues
involved in such a binding are shown, and they clearly
5
8
histidine or carnosine, activators previously investi-
gated by us and used thereafter for the design of nano-
include the histidine cluster of hCA II, comprising
residues 64, 4, 3, 10, 15 and 17. These histidine residues
(or at least some of them) should easily interact with the
highly acidic oligopeptides of the type investigated here,
and more precisely with DADD or DDDD (which
showed a high affinity only for this isozyme). The inter-
action between the positively charged imidazolium
moieties of the enzyme and the carboxylate groups of
the activators generated in this way, may explain the
high affinity of this type of CAAs for CA II, as com-
pared to their rather low affinity for CA I and bCA IV
1
3À15,17
molar CAAs.
Di-, tri- and tetrapeptides
incorporating these two amino acids, of type 8–14 on
the other hand, showed an increased affinity for the CA
active site, but important differences between the three
isozymes were detected. Thus, derivatives 8–14 showed
a rather undifferentiated affinity of 35–50 mM against
hCA I and of 43–62 mM against bCA IV, which was not
so different from the affinity showed by the two parent
amino acids, Ala and Asp. The corresponding tert-
butoxycarbonylated derivatives 15–19 did not possess at
all CA activatory properties against these two isozymes.
8
(which do not possess such a histidine cluster). Bound
at the entrance of the hCA II active site, the activators
facilitate catalysis by promoting the rate-determining
step of the catalytic cycle, that is, a proton transfer
reaction from the zinc-bound water molecule to the
environment. The proton shuttling moiety of these
CAAs is not clear at this point, but it may be the amino-
terminal group of the activator, since the Boc-deriva-
tized compounds 15–19 showed drastically reduced
activatory properties. A possible participation of the
carboxylate moieties in shuttling protons should also
Very different was the behavior of all these activators
against hCA II. Thus, the parent amino acids act as
rather weak CAAs, with activation constants of 130–
1
50 mM. Similarly weak activatory properties showed
the alanyl-alanine (AA) dipeptide 10, but not the other
di-/tri-/tetrapeptides investigated here, which all incor-
porated acidic amino acid moieties (aspartyl residues).
Thus, an almost 5-fold increase of affinity was observed
for the two dipeptides (DA and DD) as compared to the
parent amino acids, whereas the two investigated tri-
peptides (DAD and DDD) showed a further 2–3-fold
not be excluded, since the pK of these functionalities
a
when bound within the active site of the enzyme may be
quite different of those in solution.
Scozzafava, Andrea
