Chemical clamping allows for efficient phosphorylation of the RNA carrier protein Npl3.
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.
Aubol
Brandon E
BE
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0506, USA.
Ungs
Leslie
L
Lukasiewicz
Randy
R
Ghosh
Gourisankar
G
Adams
Joseph A
JA
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D009687:Nuclear Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D062367:ortho-Aminobenzoates / Q000737:chemistry
C077734:NPL3 protein, S cerevisiae; D009687:Nuclear Proteins; D010455:Peptides; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D062367:ortho-Aminobenzoates; D013912:Threonine; D012694:Serine; D000244:Adenosine Diphosphate; D012313:RNA; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate