Examination of an active‐site electrostatic node in the cAMP‐dependent protein kinase catalytic subunit

Abstract

The active site of the CAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residuesAsp 127, Glu 170, Glu 203, Glu 230, and Asp 241 -that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.Keywords: CAMP-dependent protein kinase; catalytic subunit; electrostatic interaction; pre-steady-state kinetics; substrate specificityThe catalytic subunit of CAMP-dependent protein kinase is the simplest member of the protein kinase family because it is one of the smallest and its activity is regulated by a separate subunit (R-subunit). The holoenzyme form of the enzyme is an inactive complex composed of an R-subunit homodimer and two C-subunits (Taylor et al., 1989). Active monomeric C-subunits are released after CAMP binds with high affinity to the regulatory subunits (Gill & Garren, 1969) and phosphorylate a broad range of protein substrates involved in metabolic (Krebs, 1985), transcriptional (Yamamoto et al., 1988), and signal transduction events (Cook & McCormick, 1993). Active C-subunit phosphor- ylates protein substrates on Ser and Thr residing in a common consensus sequence, Arg-Arg-X-Ser/Thr-Y, where X denotes any amino acid and Y is a hydrophobic residue (Zetterqvist et al., 1990). The P -3 and P -2 arginines3 confer 4-6 kcal/ mol of binding energy (Walsh et al., 1990) to a 20-amino acid C-subunit inhibitor (IP20). C-subunit amino acids that complement these arginines were first visualized in a crystal structure of the C-subunit complexed with IP20 (Knigh...

Dynamic architecture of a protein kinase

“…This observation is consistent with recent crystallographic, fluorescence, and computational analyses showing long-ranged effects of myristyl binding (65,66). Individual residues that have a profound effect on activity and stability [E230Q in ComF1 (67,68), W30A in ComA (69), and Y204A in ComF (70,71)] are clearly part of extended allosteric networks defined here by the community maps. Fig.…”

Kinase peptide specificity: Improved determination and relevance to protein phosphorylation

“…2). This disfavor is a logical consequence of the negatively charged catalytic cleft of PKCs, which would tend to repel substrates with negatively charged residues (11,(26)(27)(28). Second, a hydrophobic residue at the PϪ1 position is disfavored by PKC-and confers substantial selectivity compared with PKC-␦ (Fig.…”

E230Q mutation of the catalytic subunit of cAMP‐dependent protein kinase affects local structure and the binding of peptide inhibitor

“…7 The mutant is also defective in binding peptide substrates and deficient in catalyzing the phosphoryl transfer reaction. 8 Kinetic data showed that the E230Q mutation affected the binding of ATP with only 2-fold perturbation but induced a 200-fold reduction in the binding of the peptide substrate, Kemptide. 8 Another important feature of the E230Q mutant is the loss of the synergistic binding of MgATP and protein kinase inhibitor PKI (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) to the mutant.…”

“…The mutant was unable to form ternary complex crystals in the presence of MgATP and PKI. 9 However, studies on the E230Q apo-enzyme crystal structure, 9 thermostability, 8,10 and hydrogen-deuterium exchange 10 suggest that the structure of the E230Q mutant is very similar to the wild-type C-subunit. Hence, it is very likely that the altered surface electrostatic profile of the E230Q mutant causes the observed defective kinetic properties of the enzyme.…”

“…Hence, it is very likely that the altered surface electrostatic profile of the E230Q mutant causes the observed defective kinetic properties of the enzyme. 7,8,10 We applied the relaxed-complex method described by Lin et al 11 to study the effects of the E230Q mutation on PKI binding. We obtained a qualitative estimation of the probability of PKI docking to the Csubunits.…”