Kinetic Analyses of Mutations in the Glycine-Rich Loop of cAMP-Dependent Protein Kinase

Abstract

The conserved glycines in the glycine-rich loop (Leu-Gly 50 -Thr-Gly 52 -Ser-Phe-Gly 55 -ArgVal) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and presteady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s -1 in contrast to 500 s -1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the γ-phosphate of ATP. Energyminimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.Many nucleotide binding enzymes utilize glycine-rich loops that help position nucleotides for catalysis (1-3). Crystal structures of dinucleotide and mononucleotide binding enzymes define several glycine-rich loops that are distinct both in their amino acid sequence and in their orientation relative to the nucleotide. The glycine-rich loops found in dinucleotide binding enzymes [e.g., pyruvate dehydrogenase (4) and dihydrofolate reductase (5)] are typically Gly-X-Gly-X-X-Gly and provide space for the dinucleotide pyrophosphate moiety. The glycine-rich loops, or P-loops, found in mononucleotide binding enzymes [e.g., p21ras (6), adenylate kinase (7), Rec A (8), and elongation factor Tu (9)] have the consensus sequence Gly-X-X-X-X-Gly-Lys-Ser/Thr and interact with the γ-phosphate of ATP (10). In contrast, the protein kinase family of enzymes contain a glycine-rich loop with a consensus sequence Gly-X-Gly-X-X-Gly-X-Val. On the basis of numerous crystal structures, this loop is part of a -hairpin that connects -strands 1 and 2 and serves as a nucleotide-positioning loop (NPL) 1 that interacts with all three phosphates of ATP (11)(12)(13)(14)(15). The glycines in the protein kinase NPL are ...

A Novel Disulfide Bond in the SH2 Domain of the C-terminal Src Kinase Controls Catalytic Activity

“…4) which contains three highly conserved glycines that greatly control catalytic activity. [21,22] We also observed that activation of Csk through phosphopeptide binding to the SH2 domain decreases strain energies in this glycine-rich loop. [11] Likewise, in this study, activation of Csk through disulfide bond removal decreases strain in this loop.…”

Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation

“…In the crystal structure of Src (28), the side chain of Cys-277 is readily accessible, which may explain its sensitivity to oxidation. Furthermore, kinase activity appears particularly sensitive to modification to the Gly (29,30), as demonstrated for Src and Csk. All of these observations provide a mechanistic basis for the redox regulation at this residue.…”

“…The 3 Gly residues are a universally-conserved signature motif among all protein kinases, even though the intervening residues are variable. Kinetic and structural studies of both protein Ser/Thr kinase, cAMPdependent protein kinases (29), and a PTK, FPS (30), indicate that this loop is directly involved in catalysis. An examination of the amino acid sequences of all human PTKs indicates that only a small number of PTKs contain a Cys residue at the position equivalent to Cys-277 in Src (Table S1).…”

Congenital disease SNPs target lineage specific structural elements in protein kinases

“…The G-loop is one of the most flexible elements of the catalytic core and plays a key role in phosphoryl transfer. Specifically, G50 and G52 within the G-loop participate in the phosphoryl transfer reaction (16), and G55 plays a role in regulation, because it contributes to the conformational flexibility of the G-loop (17). It is notable that disease SNPs are enriched at sites involved in regulation rather than catalysis.…”