Adenosine cyclic 3',5'-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit

Cook, Paul F.; Neville, Maynard E.; Vrana, Kent E.; Hartl, Fabian; Roskoski, Robert

doi:10.1021/bi00266a011

Cited by 407 publications

(484 citation statements)

References 23 publications

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“…The steady-state kinetic parameters were measured at the maxima and minima in the plots of kobs versus total metal concentration and are listed in Tables 1 and 2. These values are consistent with previously published data for Mg2+ and Mn2+ (Armstrong et al, 1979;Cook et al, 1982). This metal ion effect on kc,, is also accompanied by tighter binding of ATP and ADP.…”

Section: "supporting

confidence: 92%

“…The secondary metal coordinates the 01 and y phosphates and the side chain of Asn 171. Extensive kinetic studies indicate that the primary metal is required for activity, while occupation of the secondary site causes a fourfold reduction in k,, when Mgz+ is the varied metal (Cook et al, 1982). A similar kinetic effect was ob- …”

Section: Discussionmentioning

confidence: 83%

“…The coupled enzyme assay designed by Cook et al (1982) worked well for all Mg2+ studies. However, significant lag phases were observed in the progress curves in the presence of either Mn2+ or Co2+.…”

Section: This Equation Is Derived From Equationmentioning

confidence: 99%

“…Comprehensive steady-state kinetic analyses reveal that the C-subunit will phosphorylate the substrate, LRRASLG, by a random kinetic mechanism, although the initial binding of ATP is preferred (Cook et al, 1982;Kong & Cook, 1988). Comparison of Kd and K,,, (Kd > K,) for LRRASLG in the presence of an ATP analog and ATP, respectively, implies that a fast step, namely phosphotransfer, follows substrate binding.…”

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confidence: 99%

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Divalent metal ions influence catalysis and active‐site accessibility in the camp‐dependent protein kinase

Adams

Taylor

1993

Protein Science

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Phosphorylation of the peptide LRRASLG by the catalytic subunit of CAMP-dependent protein kinase was measured in the presence of various divalent metals to establish the role of electrophiles in the kinetic mechanism. Under conditions of low or high metal concentrations, the apparent second-order rate constant, kc,,/Kpep,ide, and the maximal rate constant, kc,,, followed the trend Mg2+ > Co2+ > Mn2+. Competitive inhibition studies indicate that the former effect is not due to destabilization of the substrate complex, E-ATP.S. The effects of solvent viscosity on the steady-state kinetic parameters were interpreted according to a simple mechanism involving substrate binding, phosphotransfer, and product release steps and two metal chelation sites in the nucleotide pocket. Decreases in kc,, and kcol/Kpeplide result mostly from attenuations in the dissociation rate constant for ADP and the association rate constant for the substrate, respectively. Decreases in the phosphoryl transfer rate constant have only negligible to moderate effects on these parameters. The low observed values for the association rate constant of the substrate indicate that the metals control the concentration of the productive binary form, E,.ATP, and indirectly the accessibility of the active site. By comparison, Mg2+ is the best divalent metal catalyst because it uniformly lowers the transition state energies for all steps in the kinetic mechanism, permitting maximum flux of substrate to product. The data suggest that CAMP-dependent protein kinase uses metal ions to serve multiple roles in facilitating phosphotransfer and accelerating substrate association and product dissociation.Keywords: catalytic subunit; divalent metal ions; solvent viscosity; steady-state kinetics Many theories have been proposed to explain the large rate enhancements by metal catalysts at the active sites of enzymes. Data attained from enzymatic and model studies suggest that metal ions may accelerate reactions by lowering the chemical transition state energy through polarization of the electrophile, charge reduction, and stabilization of the leaving group (Jencks, 1987). In addition, ions may confer tight binding or proper orientation of the substrate's functional groups. An efficient enzyme recognizes substrate, easily modifies it, and then quickly releases the product without populating unproductive or very stable intermediate species. Enzymes accomplish this by lowering the transition state energies of the chemical and conformational steps and equalizing the ground states

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Section: "supporting

confidence: 92%

Section: Discussionmentioning

confidence: 83%

Section: This Equation Is Derived From Equationmentioning

confidence: 99%

mentioning

confidence: 99%

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Divalent metal ions influence catalysis and active‐site accessibility in the camp‐dependent protein kinase

Adams

Taylor

1993

Protein Science

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“…Kinetic studies predict a preferred, but not obligatory, ordered binding of substrates for the C-subunit with ATP binding preceding substrate binding (Cook et al, 1982;Whitehouse et al, 1983). ATP binds in a deep pocket, and in the ternary complex, closed structure is completely buried by the inhibitor peptide, &strands l and 2, and the C-terminal tail (Zheng et al, 1993a).…”

Section: Ordered Binding Of Substratesmentioning

confidence: 99%

Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations

et al. 1993

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Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of CAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32,2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.

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Protein Kinase Structure, Function, and Regulation

Matthews¹,

Gerritsen²

2010

Targeting Protein Kinases for Cancer Therapy

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Adenosine cyclic 3',5'-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit

Cited by 407 publications

References 23 publications

Divalent metal ions influence catalysis and active‐site accessibility in the camp‐dependent protein kinase

Divalent metal ions influence catalysis and active‐site accessibility in the camp‐dependent protein kinase

Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations

Protein Kinase Structure, Function, and Regulation

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