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

The glycine-rich loop, one of the most important motifs in the conserved pr otein kinase catalytic core, embraces the entire nucleotide, is very mobile , and is exquisitely sensitive to what occupies the active site deft. Of th e three conserved glycines [G(50)TG(52)SFG(55) in cAMP-dependent protein ki nase (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 t he 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 sugge sts that Ser53 also might be essential for stabilization of the peptide sub strate-enzyme complex via a hydrogen bond between the P-site carbonyl in PK I and the Ser53 side-chain hydroxyl [Bossemeyer, D. et al. (1993) EMBO J. 1 2, 849]. To address the importance of the Ser53 side chain in catalysis, in hibition, and P-site specificity, Ser53 was replaced with threonine, glycin e, and proline. Removal of the side chain (i.e., mutation to glycine) had n o effect on the steady-state phosphorylation of a peptide substrate (LRRASL G) or on the interaction with physiological inhibitors, including the type- I and -II regulatory subunits and PKI. However, this mutation did affect th e P-sire specificity; the glycine mutant can more readily phosphorylate a P -site threonine in a peptide substrate (5-6-fold better than wild-type). Th e proline mutant is compromised catalytically with altered k(cat) and K-m f or both peptide and ATP and with altered sensitivity to both regulatory sub units and PKI. Steric constraints as well as restricted flexibility could a ccount for these effects. These combined results demonstrate that while the backbone amide of Sers' may be required for efficient catalysis, the side chain is not.