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(Francis et al. 2006 ). Thus, it remains puzzling whether the second metal ion is
magnesium or manganese in biological systems and whether different PDE families
prefer different divalent metals.
The structure-based sequence alignment reveals the conservation of residues in
the active site of PDE families (Table 1 ). In addition to the four invariant metal-
binding residues, three residues at the active sites are invariant across PDE
families (His160, His204, and Gln369 in PDE4D2). His160 was proposed to
serve as a general acid for the catalysis (Huai et al. 2003 ; Liu et al. 2008 ),
while the exact role of His204 remains to be identified. In the nucleotide-binding
pocket, the invariant glutamine (Gln369 in PDE4D2, Table 1 ) forms at least one
hydrogen bond with substrates or inhibitors, and a conserved phenylalanine
(tryptophan in PDE11) stacks against substrates and inhibitors (Fig. 2 ). These
interactions are two characteristics for binding of substrates or inhibitors in all
PDE families.
3 Substrate Specificity
After the first report of the isolation of the cAMP-specific PDE from beef heart
(Butcher and Sutherland 1962 ), various isoforms of PDEs were discovered to have
different capabilities for hydrolyzing cAMP and cGMP (Beavo et al. 1970 ; Monn
and Christiansen 1971 ). For over 48 years, the essential characteristics of the
mechanism whereby the conserved catalytic domains of PDE families selectively
recognize the subtle differences between cAMP and cGMP have remained elusive.
In kinetic theory, the substrate specificity is quantitatively defined by the ratio of the
specific constants of one substrate versus another, specifically by (k cat /K M ) cAMP
over (k cat /K M ) cGMP for PDEs. However, since the pure PDEs, and thus the k cat
values, were not easily obtained in early times, the apparent equilibrium constant
K M has been conveniently used to represent the substrate specificity of PDEs
(Mehats et al. 2002 ). In recent years, due to great progress on the expression and
purification of high quality recombinant PDEs, the k cat values and the specific
constants can now be measured, and thus the substrate specificity can be fully
defined. As shown in Table 2 , the K M is indeed dominant in determination of the
substrate specificity. However, caution should be taken for evaluation of the para-
meters, respectively, obtained from the catalytic domain and full-length enzymes.
Although several PDE families show similar kinetic parameters for the catalytic
domains and the full-length enzymes, the allosteric modes of certain PDE families
may have different K M and k cat of these enzymes. In addition, the potential
dimerization of some highly purified PDEs may offer the possibility of alterations
in k cat , relative to a monomer. For example, the PDE4 model suggests that a long-
form dimer may have an activity that is half that of the dimer due to capping of one
of the active site in one partner by a regulatory portion of the other partner (Burgin
et al. 2010 ).
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