Kinetic mechanistic studies of Cdk5/p25-catalyzed H1P phosphorylation: metal effect and solvent kinetic isotope effect.
Cdk5/p25 is a member of the cyclin-dependent, Ser/Thr kinase family and has been identified as one of the principle Alzheimer's disease-associated kinases that promote the formation of hyperphosphorylated tau, the major component of neurofibrillary tangles. We and others have been developing inhibitors of cdk5/p25 as possible therapeutic agents for Alzheimer's disease (AD). In support of these efforts, we examine the metal effect and solvent kinetic isotope effect on cdk5/p25-catalyzed H1P (a histone H-1-derived peptide) phosphorylation. Here, we report that a second Mg(2+) in addition to the one forming the MgATP complex is required to bind to cdk5/p25 for its catalytic activity. It activates cdk5/p25 by demonstrating an increase in k(cat) and induces a conformational change that favors ATP binding but has no effect on the binding affinity for the H1P peptide substrate. The binding of the second Mg(2+) does not change the binding order of substrates. The reaction follows the same rapid equilibrium random mechanism in the presence or absence of the second Mg(2+) as evidenced by initial velocity analysis and substrate analogue and product inhibition studies. A linear proton inventory with a normal SKIE of 2.0 +/- 0.1 in the presence of the second Mg(2+) was revealed and suggested a single proton transfer in the rate-limiting phosphoryl transfer step. The pH profile revealed a residue with a pK(a) of 6.5 that is most likely the general acid-base catalyst facilitating the proton transfer.
Liu
Min
M
Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, 65 Landsdowne Street, Fourth Floor, Cambridge, Massachusetts 02139, USA. mliu@rics.bwh.harvard.edu
Girma
Eleni
E
Glicksman
Marcie A
MA
Stein
Ross L
RL
D000244:Adenosine Diphosphate / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D051360:Cyclin-Dependent Kinase 5 / Q000737:chemistry / Q000378:metabolism*; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011522:Protons; D012997:Solvents; D013379:Substrate Specificity
D006657:Histones; D009419:Nerve Tissue Proteins; D011522:Protons; D012997:Solvents; C118136:TPPP protein, human; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; D008274:Magnesium