Nuclear magnetic resonance studies of the conformation and kinetics of the peptide-substrate at the active site of bovine heart protein kinase

Granot, Joseph; Mildvan, Albert S.; Bramson, H. Neal; Thomas, Nancy E.; Kaiser, E. T.

doi:10.1021/bi00506a024

Cited by 52 publications

(46 citation statements)

References 35 publications

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“…Strict steric constraints at the P ϩ1 site are also indicated by Kemptide analog studies. Substitution of the P ϩ1 residue in Kemptide with Pro (LRRASPG) resulted in a significant reduction in substrate capacity (56), but introduction of N ␣ -methyl Leu in place of Leu at the P ϩ1 position resulted in only a moderate loss of substrate capacity (57).…”

Section: Inhibition Of C Subunit By Cimp-saturated Ri Subunitsmentioning

confidence: 99%

Identification of Critical Determinants for Autoinhibition in the Pseudosubstrate Region of Type Iα cAMP-dependent Protein Kinase

Poteet‐Smith

Shabb

Francis

et al. 1997

Journal of Biological Chemistry

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The consensus substrate site for cAMP-dependent protein kinase (PKA) is Arg-Arg-Xaa-Ser(P)-Xaa and the autoinhibitory domain of the PKA type I alpha regulatory subunit (RI subunit) contains a similar sequence, Arg92-Arg-Arg-Arg-Gly-Ala-Ile-Ser-Ala-Glu. The italicized amino acids form a putative pseudosubstrate site (Ser is replaced with Ala), which together with adjacent residues could competitively inhibit substrate phosphorylation by the PKA catalytic subunit (C subunit). The present studies determine the contributions of Arg92-95, Ile98, and Glu101 to inhibitory potency. Amino-terminal truncation of RI subunit through Arg92 (delta1-92) or Arg93 (delta1-93) had no detectable effect on inhibition of C subunit. Truncation through Arg94 (delta1-94), or point mutation of Arg95 within truncated mutants (delta1-93.R95A or delta1-92.R95A), caused a dramatic reduction in inhibitory potency. Truncation through Arg95 (delta1-95) had a greater effect than did replacement or deletion of Arg94 or Arg95 alone. Using full-length RI subunit, the inhibitory potency was reduced by replacing Ile98 with Ala, Gly, or Gln, but not by replacing it with Val. The inhibitory potency of RI subunit was unchanged when Glu101 was replaced with Ala or Gln. It is concluded that Arg94, Arg95 and, to a lesser extent, Ile98 are vital constituents of PKA autoinhibition by type I alpha R subunit.

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Section: Inhibition Of C Subunit By Cimp-saturated Ri Subunitsmentioning

confidence: 99%

Identification of Critical Determinants for Autoinhibition in the Pseudosubstrate Region of Type Iα cAMP-dependent Protein Kinase

Poteet‐Smith

Shabb

Francis

et al. 1997

Journal of Biological Chemistry

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“…This enzyme is readily purified in milligram quantities, and a synthetic peptide substrate (kemptide) is available, which is phosphorylated with favorable kinetic parameters (10). The conformation of this peptide bound to the active site of cAMP-dependent protein kinase has been elucidated (4,5).…”

Section: Discussionmentioning

confidence: 99%

“…The conformation of a peptide substrate bound to the enzyme has been deduced from NMR measurements (4,5). Several investigators have used affinity labels to study the regulatory and catalytic subunits of the enzyme.…”

mentioning

confidence: 99%

Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label.

Miller

Kaiser

1988

Proc. Natl. Acad. Sci. U.S.A.

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A peptide-based photoaffinity label for the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)]. By using solid-phase peptide synthesis methodology, DL-Phe(pBz) was incorporated into the cAMP-dependent protein kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in place of the phosphorylatable serine. The diastereomeric peptides were separated by reverse-phase HPLC. The peptide substrate analog containing L-Phe(pBz) had a Ki of approximately 110 IAM at pH 7.5. When photolyzed at 350 nm in the presence of the enzyme, this peptide caused time-and concentration-dependent inactivation. Radioactive acetylated L-Phe-(pBz) peptide was used to establish the binding stoichiometry of peptide to enzyme; these results, together with protection experiments, showed the photoaffinity labeling to be specific (~1:1). To identify the residues that were modified on the catalytic subunit, the photoinactivated enzyme was cleaved with CNBr and V8 protease (Staphylococcus aureus). The resulting peptide fragments were purified by HPLC and were sequenced; these experiments identified the modified residues as Gly-125 and Met-127. This region of the cAMP-dependent protein kinase catalytic subunit contains many residues that are conserved in serine-and tyrosine-protein kinases.The cAMP-dependent protein kinase is a central enzyme in eukaryotic metabolism and is believed to be responsible for all of the physiological effects of cAMP (1). The inactive holoenzyme contains two regulatory and two catalytic subunits. Upon binding of cAMP, the holoenzyme dissociates to give two catalytic subunits and a regulatory dimer (2). The catalytic subunit catalyzes the transfer of the y-phosphoryl group of ATP to appropriate protein or peptide substrates.The primary sequence of the catalytic subunit has been determined (3), but the structure of the enzyme active site remains unclear. The conformation of a peptide substrate bound to the enzyme has been deduced from NMR measurements (4, 5). Several investigators have used affinity labels to study the regulatory and catalytic subunits of the enzyme. The catalytic subunits from bovine heart (6) and porcine skeletal muscle (7) have been modified with the reagent 5'-(p-fluorosulfonylbenzoyl)adenosine; this label was shown to modify Lys-72 specifically (8). Bramson et al. (9) modified Cys-199 with the peptide Leu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly and concluded that this residue was in the enzyme active site. This experiment demonstrated the feasibility of using analogs of kemptide (Leu-Arg-ArgAla-Ser-Leu-Gly), a good substrate of the enzyme (10), to modify the peptide binding site. Peptides containing N8-bromoacetylornithine have been used to alkylate ; M. Doughty and E.T.K., unpublished results). This paper describes the synthesis of a photoaffinity label designed to modify the peptide binding site of the catalytic subunit. The amino acid p-benzoyl-L-phenylalanine [LPhe(pBz)] was chosen as the photoactive probe becaus...

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“…In addition, a few kinases have been shown to bind a second divalent cation that may be either activating as with pyruvate kinase (7,8) or inhibiting as with cAMP dependent protein kinase (9)• It is the presence of this pair of cations at the active sites of these latter two enzymes that has allowed detailed NMR studies to be carried out and has aided in the formulation of detailed working models of their active-site geometries (10)(11)(12)(13)• These studies will be discussed elsewhere in the chapter. This compound has been found to be an alternate substrate, albeit a rather poor one, for several kinases (pyruvate kinase, hexokinase, phosphofructokinase, and adenylate kinase) (22,23).…”

Section: For All Kinases One Divalent Cation Is Nucleotide Boundmentioning

confidence: 99%

Design of Kinase Inhibitors

Kenyon

Reddick

1984

ACS Symposium Series

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Nuclear magnetic resonance studies of the conformation and kinetics of the peptide-substrate at the active site of bovine heart protein kinase

Cited by 52 publications

References 35 publications

Identification of Critical Determinants for Autoinhibition in the Pseudosubstrate Region of Type Iα cAMP-dependent Protein Kinase

Identification of Critical Determinants for Autoinhibition in the Pseudosubstrate Region of Type Iα cAMP-dependent Protein Kinase

Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label.

Design of Kinase Inhibitors

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