Serine-53 at the Tip of the Glycine-Rich Loop of cAMP-Dependent Protein Kinase:  Role in Catalysis, P-Site Specificity, and Interaction with Inhibitors

Abstract

The glycine-rich loop, one of the most important motifs in the conserved protein kinase catalytic core, embraces the entire nucleotide, is very mobile, and is exquisitely sensitive to what occupies the active site cleft. Of the three conserved glycines [G(50)TG(52)SFG(55) in cAMP-dependent protein kinase (cAPK)], Gly(52) is the most important for catalysis because it allows the backbone amide of Ser(53) at the tip of the loop to hydrogen bond to the gamma-phosphate of ATP [Grant, B. D. et al. (1998) Biochemistry 37, 7708]. The structural model of the catalytic subunit:ATP:PKI((5)(-)(24)) (heat-stable protein kinase inhibitor) ternary complex in the closed conformation suggests that Ser(53) also might be essential for stabilization of the peptide substrate-enzyme complex via a hydrogen bond between the P-site carbonyl in PKI and the Ser(53) side-chain hydroxyl [Bossemeyer, D. et al. (1993) EMBO J. 12, 849]. To address the importance of the Ser(53) side chain in catalysis, inhibition, and P-site specificity, Ser(53) was replaced with threonine, glycine, and proline. Removal of the side chain (i.e., mutation to glycine) had no effect on the steady-state phosphorylation of a peptide substrate (LRRASLG) or on the interaction with physiological inhibitors, including the type-I and -II regulatory subunits and PKI. However, this mutation did affect the P-site specificity; the glycine mutant can more readily phosphorylate a P-site threonine in a peptide substrate (5-6-fold better than wild-type). The proline mutant is compromised catalytically with altered k(cat) and K(m) for both peptide and ATP and with altered sensitivity to both regulatory subunits and PKI. Steric constraints as well as restricted flexibility could account for these effects. These combined results demonstrate that while the backbone amide of Ser(53) may be required for efficient catalysis, the side chain is not.

Investigation of Invariant Serine/Threonine Residues in Mevalonate Kinase

“…These data for the glycine-rich, serine-containing motif derive additional significance upon extrapolation to other members of the GHMP kinase family of phosphotransferases. The 4000-fold impact on V max upon elimination of the Ser-146 hydroxyl group sharply contrasts with observations made with protein kinase A, where elimination of the ATP binding loop's serine hydroxyl has no significant effect (24). Instead, the amide backbone of the loop residue makes the important interaction with ATP.…”

Characterization of the Mechanisms Controlling Greatwall Activity

“…1A). This domain covers and anchors the nontransferable ATP ␣/␤-phosphates and leaves the ␥-phosphate solvent exposed, positioning the ATP appropriately for ␥-phosphate transfer to the substrate (1,18). In order to analyze the activity of this mutant we performed an in vitro kinase assay using myelin basic protein (MBP) as a substrate.…”

Activating PRKACB somatic mutation in cortisol-producing adenomas

“…The particular function of Ser54 has been previously investigated (11). This residue is located at the tip of the G-loop, which is part of the small amino-terminal lobe of the C subunit.…”