Abstract
A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both Km and kcat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in Km (ATP), an 8-12-fold increase in Km (peptide), and a 3-5-fold drop in kcat. The Km (ATP) and Km (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the kcat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.
Highlights
A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases
All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family
Conserved in this core are the amino acids involved in nucleotide binding and catalysis with one of the most highly conserved features being a GXGXXG motif near the amino terminus
Summary
A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. The eukaryotic protein kinases specific for serine, threonine, and tyrosine all share a conserved catalytic core that folds into a topologically similar three-dimensional structure (2–5) Conserved in this core are the amino acids involved in nucleotide binding and catalysis with one of the most highly conserved features being a GXGXXG motif near the amino terminus. The glycine-rich sequence is located in the small lobe; it is part of a -strand-loop--strand nucleotide positioning motif (Fig. 1) that is an integral part of the ATP-binding site. Because the phosphoryl transfer step is 20-fold faster than the kcat (21), localized mobility of the glycine-rich loop is likely to be of great functional importance both for cAPK and for protein kinases, in general
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