Abstract
Protein kinases phosphorylate the appropriate protein substrate by recognizing residues both proximal and distal to the site of phosphorylation. Although these distal contacts may provide excellent binding affinities (low Km values) through stabilization of the enzyme-substrate complex, these contacts could reduce catalytic turnover (decrease kcat) through slow phosphoprotein release. To investigate how protein kinases can overcome this problem and maintain both high substrate affinities and high turnover rates, the phosphorylation of the yeast RNA transport protein Npl3 by its natural protein kinase, Sky1p, was evaluated. Sky1p bound and phosphorylated Npl3 with a Km that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants. Surprisingly, this extraordinary difference is not the result of high affinity Npl3 binding. Rather, Npl3 achieves a low Km through a rapid and favorable phosphoryl transfer step. This step serves as a chemical clamp that locks the protein substrate in the active site without unduly stabilizing the product phosphoprotein and slowing its release. The chemical clamping mechanism offers an efficient means whereby a protein kinase can simultaneously achieve both high turnover and good substrate binding properties.
Highlights
The phosphorylation of hydroxyl-containing amino acids in eukaryotic proteins controls numerous physiological responses essential for cell function
Much is known about the protein kinase family on the structural and functional levels [3,4,5,6], less is known about the interactions of these enzymes with their physiological protein substrates
The catalytic efficiency of phosphorylating the protein substrate MAPKAP2 using p38 MAPK1 is ϳ2 orders of magnitude higher than that for a 14-residue peptide based on the consensus sequence [10]
Summary
The phosphorylation of hydroxyl-containing amino acids (i.e. serine, threonine, and tyrosine) in eukaryotic proteins controls numerous physiological responses essential for cell function. Csk phosphorylates the protein substrate Lck Ͼ3 orders of magnitude more efficiently than a 9-residue peptide based on the phosphorylation site [11] Studies of this type support a model in which regions beyond the limited consensus sequence interact with the protein kinase scaffold and govern high efficiency protein phosphorylation. It has been shown that the high affinity interaction between Csk and its substrate Src relies on a specific docking region in helix ␣D of the kinase domain, a secondary structural element outside the catalytic cleft [15] These examples highlight discrete surfaces within the kinase domain, other studies reveal that substrate-binding sites can lie on non-catalytic regulatory proteins. The phosphoryl transfer step functions as a chemical clamp that provides exquisite selectivity, but avoids the dilemma of over-stabilizing the phosphoproduct and limiting the catalytic cycle through slow phosphoprotein release
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