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The catalytic loop is the region of divergence between Ser/Thr and Tyr kinases. In cAPK and all Ser/Thr
Kinases, Lys168 interacts with the γ phosphate of ATP during catalysis [12]. The role of Lys is replaced
by Arg [9] and the insulin receptor tyrosine kinase structure [3] shows Arg1136 in a similar position as
Lys168 in the active site of cAPK.
The catalytic loop and β-strand 7 are followed by β-strand 8 and a short DFG conserved motif. This
conserved motif consists of invariant Asp184, a ligand to the metal site, and invariant Phe185. The DFG
motif is followed by β-strand 9, an activation loop that includes Thr197. The activation loop differs
considerably among unphosphorylated kinases, CDK-2, ERK-2, and insulin receptor kinase.
Furthermore, two crystal structures, twitchin protein kinase [2] and phosphorylase kinase [5], both lack a
phosphorylation site in this region. In the first structure, twitchin protein kinase, Thr197 and Arg165, are
replaced by hydrophobic residues, Val6098 and Leu6062 respectively [2]. This was predicted using
modeling [18] and subsequently confirmed by the three-dimensional structure. In the second structure,
phosphorylase kinase, the Thr197 is replaced by Glu182, which interacts with Arg148 [5]. Hence, in
both structures the regulatory function of the phosphorylation site is replaced by a stable scaffold
secured by either hydrophobic or electrostatic interactions. Since it has been shown that this loop
provides a stable template for PKI(5–24) binding in cAPK, the status of phosphorylation of the
activation loop critically affects the substrate binding. This is demonstrated in c-Src, a homolog of the
Rous Sarcoma virus oncogene by mutation of Arg385, which is predicted to interact with the phosphate
of Tyr416, and results in loss of activity toward the exogenous substrate [19].
The activation loop is followed by a P+1 loop which accommodates the P+1 site of the substrate. The
P+1 loop is followed by invariant Glu208, which forms a salt bridge with invariant Arg280. This
conserved pair plays a structural role and as the structure of CK-1 [16] shows, it can be replaced by
other charged residues that maintain the same fold of the lower lobe. The P+1 loop and Glu 208 are
followed by helix F consisting of invariant Asp220, followed by helices G, H, and I. The helix J and the
C-terminal tail of cAPK, which are absent in other protein kinases, undergo a large motion during the
cleft opening.
The opening of the cleft results in loss of hydrogen bonds provided by the γ phosphate of ATP and the
peptide that would bridge the lower and upper lobe of the enzyme. The motion of the upper domain
increases the accessibility of the ATP binding site and one can envision that in the “open” conformation
ATP binds. Yet, in the “closed” conformation ATP and its γ phosphate are positioned for a nucleophilic
attack on the substrate. The motion of this lobe—which includes His87, one of the ligands to Thr197
[15]—indicates that the phosphorylation of this site will be important for conformational diversity of the
upper lobe. This is confirmed by the varying degrees of displacement of the upper lobe
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