Characterization of PKACα enzyme kinetics and inhibition in an HPLC assay with a chromophoric substrate

Abstract

Here we describe a convenient, inexpensive, and non-hazardous method for the measurement of kinase activity of the catalytic subunit of cAMP-dependent protein kinase (PKACα). The assay is based on the separation of a substrate peptide labeled with a strong chromophore from the phosphorylated product peptide by high-performance liquid chromatograph (HPLC) and quantification of the product ratiometrically at a wavelength in the visual spectrum (Vis). The utility and reliability of the HPLC-Vis assay were demonstrated by characterizing the kinetic parameters (KM, Vmax) of the new Rh-MAB-Kemptide substrate, a commercially prepared TAMRA-Kemptide substrate, and ATP as well as the potency (IC50, Ki) of the known PKACα inhibitors H89 and PKI(5-24). The advantages of this assay are that it is convenient and inexpensive, uses readily synthesized or commercially available substrates that are shelf-stable, uses a common piece of laboratory equipment, and does not require any hazardous materials such as radioactive γ-32P-ATP. The assay format is also highly flexible and could be adapted for testing of many different kinases by changing the peptide substrate sequence.

Kinetics and inhibition studies of the L205R mutant of cAMP‐dependent protein kinase involved in Cushing's syndrome

“…The fragment containing R205 was identified ( 195 TWTLCGTPEYR 205 , calculated: 1327.49 Da; found: 1327.16 Da), confirming installation of the mutation. wt-PKACa was produced and purified as previously reported [11] in parallel with the L205R-PKACa mutant.…”

“…Kinetics assays were performed using an HPLC-Vis assay with a rhodamine-labeled Kemptide analogue (Rh-MAB-Kemptide), which we have reported elsewhere [11]. Assays were performed in 96-well plates at 25°C with a total reaction volume of 200 lL.…”

“…Three experimental replicates were carried out for each curve, and the mean and SEM for the IC 50 and K i for each inhibitor were determined. For comparison, wt-PKACa replicates were performed in parallel on the same 96-well plates with technical and experimental replicates for the L205R mutant as reported elsewhere [11].…”

“…1A), an important site for substrate, regulatory subunit, and inhibitor recognition. The hydrophobic side chain of Ile22 of PKI (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) binds in the P+1-binding site, creating a positive binding interaction and contributing to overall inhibitor binding. In the cocrystal structure of the L205R-PKACa mutant with PKI(5-24) and ATP ( Fig.…”

“…1B) [10], the mutation of Leu205 to Arg results in the larger Arg205 residue occupying the P + 1 pocket (now defined as Leu198, Gly200, Pro202, and Arg205), the binding pocket normally occupied by Ile22 of PKI. The L205R mutation prevents Ile22 of PKI from binding in the P + 1 pocket and causes a change in binding conformation of the C-terminal end of PKI (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The mutant structure offers an explanation for the lack of binding by the regulatory subunits and predicts a lack of inhibition of L205R-PKACa by PKI, as both of these require a hydrophobic binding interaction in the P + 1 pocket of wt-PKACa for regulation or inhibition.…”