PMID- 10400656
OWN - NLM
STAT- MEDLINE
DCOM- 19990819
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 29
DP  - 1999 Jul 16
TI  - Kinetic analysis of human serine/threonine protein phosphatase 2Calpha.
PG  - 20336-43
AB  - The PPM family of Ser/Thr protein phosphatases have recently been shown to
      down-regulate the stress response pathways in eukaryotes. Within the stress
      pathway, key signaling kinases, which are activated by protein phosphorylation,
      have been proposed as the in vivo substrates of PP2C, the prototypical member of 
      the PPM family. Although it is known that these phosphatases require metal
      cations for activity, the molecular details of these important reactions have not
      been established. Therefore, here we report a detailed biochemical study to
      elucidate the kinetic and chemical mechanism of PP2Calpha. Steady-state kinetic
      and product inhibition studies revealed that PP2Calpha employs an ordered
      sequential mechanism, where the metal cations bind before phosphorylated
      substrate, and phosphate is the last product to be released. The metal-dependent 
      activity of PP2C (as reflected in kcat and kcat/Km), indicated that Fe2+ was
      1000-fold better than Mg2+. The pH rate profiles revealed two ionizations
      critical for catalytic activity. An enzyme ionization with a pKa value of 7 must 
      be unprotonated for catalysis, and an enzyme ionization with a pKa of 9 must be
      protonated for substrate binding. Bronsted analysis of substrate leaving group
      pKa indicated that phosphomonoester hydrolysis is rate-limiting at pH 7. 0, but
      not at pH 8.5 where a common step independent of the nature of the substrate and 
      alcohol product limits turnover (kcat). Rapid reaction kinetics between
      phosphomonoester and PP2C yielded exponential "bursts" of product formation,
      consistent with phosphate release being the slow catalytic step at pH 8.5.
      Dephosphorylation of synthetic phosphopeptides corresponding to several protein
      kinases revealed that PP2C displays a strong preference for diphosphorylated
      peptides in which the phosphorylated residues are in close proximity.
FAU - Fjeld, C C
AU  - Fjeld CC
AD  - Department of Biochemistry and Molecular Biology, Oregon Health Sciences
      University, Portland, OR 97201-3098, USA.
FAU - Denu, J M
AU  - Denu JM
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Phosphopeptides)
RN  - 0 (Saccharomyces cerevisiae Proteins)
RN  - 42Z2K6ZL8P (Manganese)
RN  - EC 3.1.3.16 (PTC1 protein, S cerevisiae)
RN  - EC 3.1.3.16 (Phosphoprotein Phosphatases)
RN  - EC 3.1.3.16 (Protein Phosphatase 2)
RN  - EC 3.1.3.16 (Protein Phosphatase 2C)
RN  - I38ZP9992A (Magnesium)
SB  - IM
MH  - Amino Acid Sequence
MH  - Catalysis
MH  - Humans
MH  - Hydrogen-Ion Concentration
MH  - Kinetics
MH  - Magnesium/metabolism
MH  - Manganese/metabolism
MH  - Phosphopeptides/chemistry/metabolism
MH  - Phosphoprotein Phosphatases/*metabolism
MH  - Protein Phosphatase 2
MH  - Protein Phosphatase 2C
MH  - *Saccharomyces cerevisiae Proteins
MH  - Substrate Specificity
EDAT- 1999/07/10 00:00
MHDA- 1999/07/10 00:01
CRDT- 1999/07/10 00:00
PHST- 1999/07/10 00:00 [pubmed]
PHST- 1999/07/10 00:01 [medline]
PHST- 1999/07/10 00:00 [entrez]
AID - 10.1074/jbc.274.29.20336 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Jul 16;274(29):20336-43. doi: 10.1074/jbc.274.29.20336.