PMID- 19719323
OWN - NLM
STAT- MEDLINE
DCOM- 20091203
LR  - 20211020
IS  - 1520-4995 (Electronic)
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 48
IP  - 40
DP  - 2009 Oct 13
TI  - Functional energetic landscape in the allosteric regulation of muscle pyruvate 
      kinase. 2. Fluorescence study.
PG  - 9456-65
LID - 10.1021/bi900280u [doi]
AB  - The energetic landscape of the allosteric regulatory mechanism of rabbit muscle 
      pyruvate kinase (RMPK) was characterized by isothermal titration calorimetry 
      (ITC). Four novel insights were uncovered. (1) ADP exhibits a dual property. 
      Depending on the temperature, ADP can regulate RMPK activity by switching the 
      enzyme to either the R or T state. (2) The assumption that ligand binding to RMPK 
      is state-dependent is only correct for PEP but not Phe and ADP. (3) The effect of 
      pH on the regulatory behavior of RMPK is partly due to the complex pattern of 
      proton release or absorption linked to the multiple linked equilibria which 
      govern the activity of the enzyme. (4) The R <--> T equilibrium is accompanied by 
      a significant DeltaC(p), rendering RMPK most sensitive to temperature under 
      physiological conditions. To rigorously test the validity of conclusions derived 
      from the ITC data, in this study a fluorescence approach, albeit indirect, that 
      tracks continuous structural perturbations was employed. Intrinsic Trp 
      fluorescence of RMPK in the absence and presence of substrates 
      phosphoenolpyruvate (PEP) and ADP, and the allosteric inhibitor Phe, was measured 
      in the temperature range between 4 and 45 degrees C. For data analysis, the 
      fluorescence data were complemented by ITC experiments to yield an extended data 
      set allowing more complete characterization of the RMPK regulatory mechanism. 
      Twenty-one thermodynamic parameters were derived to define the network of linked 
      interactions involved in regulating the allosteric behavior of RMPK through 
      global analysis of the ITC and fluorescent data sets. In this study, 27 
      independent curves with more than 1600 experimental points were globally 
      analyzed. Consequently, the consensus results substantiate not only the 
      conclusions derived from the ITC data but also structural information 
      characterizing the transition between the active and inactive states of RMPK and 
      the antagonism between ADP and Phe binding. The latter observation reveals a 
      novel role for ADP in the allosteric regulation of RMPK.
FAU - Herman, Petr
AU  - Herman P
AD  - Faculty of Mathematics and Physics, Institute of Physics, Charles University, Ke 
      Karlovu 5, 121 16 Prague, Czech Republic. herman@karlov.mff.cuni.cz
FAU - Lee, J Ching
AU  - Lee JC
LA  - eng
GR  - R01 GM077551/GM/NIGMS NIH HHS/United States
GR  - R01 GM077551-04/GM/NIGMS NIH HHS/United States
GR  - GM77551/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Ligands)
RN  - 47E5O17Y3R (Phenylalanine)
RN  - 61D2G4IYVH (Adenosine Diphosphate)
RN  - 73-89-2 (Phosphoenolpyruvate)
RN  - 8DUH1N11BX (Tryptophan)
RN  - EC 2.7.1.40 (Pyruvate Kinase)
SB  - IM
MH  - Adenosine Diphosphate/chemistry
MH  - Allosteric Regulation
MH  - Animals
MH  - Energy Metabolism
MH  - Entropy
MH  - Enzyme Activation
MH  - Ligands
MH  - Models, Chemical
MH  - Muscle, Skeletal/*enzymology
MH  - Phenylalanine/chemistry
MH  - Phosphoenolpyruvate/chemistry
MH  - Protein Binding
MH  - Protein Structure, Tertiary
MH  - Pyruvate Kinase/antagonists & inhibitors/*chemistry/*metabolism/physiology
MH  - Rabbits
MH  - *Spectrometry, Fluorescence/methods
MH  - Tryptophan/chemistry/metabolism
PMC - PMC2772997
MID - NIHMS146297
EDAT- 2009/09/02 06:00
MHDA- 2009/12/16 06:00
PMCR- 2010/10/13
CRDT- 2009/09/02 09:00
PHST- 2009/09/02 09:00 [entrez]
PHST- 2009/09/02 06:00 [pubmed]
PHST- 2009/12/16 06:00 [medline]
PHST- 2010/10/13 00:00 [pmc-release]
AID - 10.1021/bi900280u [doi]
PST - ppublish
SO  - Biochemistry. 2009 Oct 13;48(40):9456-65. doi: 10.1021/bi900280u.