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site coordinated by invariant Asn171 is also sequestered by the α and γ phosphates. The residue Lys168
is at hydrogen-bond distance from the γ phosphate. The postulated in-line mechanism of
phosphotransfer in cAPK [23] can be examined through an analysis of the MnATP and PKI (5–24), Ser
substrate peptide and ADP complexes [24]. A comparison between cAPK and IRK structures indicates
that Ser versus Tyr specificity is obtained by displacement of the substrate binding site in such a way
that the hydroxyl of nucleophiles of both Ser and Tyr fall in the same point in the active site facing
corresponding catalytic bases Asp1132 and Asp166 for IRK and cAPK respectively.
The purine base of ATP is anchored to the enzyme by three hydrogen bonds, two of them involve the 6amino
group and N1 nitrogen, which interact with the backbone atoms of Glu121 and Val123. the 6amino
group and N1 nitrogen of the purine base form the hydrogen bonds also in the structure of CK-1
and in the structure of phosphorylase kinase. Yet, in the structure of inactive CDK-2, the purine base
forms only one hydrogen bond via its N6 position. While N7 nitrogen interacts directly in cAPK with
the side chain of Thr183 in CK-1, this nitrogen interacts directly with Glu55 and Tyr59 via two
hydrogen-bonded water molecules and in phosposhorylase kinase via one water molecule. Hence, the
N7 nitrogen and its interaction with the enzyme is a region for potential modification of ATP
competitive inhibitors. Ribose is held by both enzyme (Glu127 and Glu170) and inhibitor (P-3 Arg).
While the side chain of Glu127 interacts with 2'-OH of ribose in cAPK, in CK-1 the 2'-OH interacts via
two water molecules with Ser91 and Asp94. In ERK-2, 2'-OH interacts with Asp109. This region has
been utilized to design ATP-based specific inhibitors by modifications of the ATP-competitive
nonspecific inhibitor staurosporine [25], see Figure 4.
In the model cAPK with bound staurosporine inhibitor, the lactam amide group of the inhibitor functions
as a bidentate hydrogen bond donor-acceptor
Figure 4
Chemical structures of staurosporine inhibitor, left, and
CGP52411.
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