The Phosphorylation Potential
Determination and Uses in Disease
Richard L Veech, MD DPhil
Lab of Metabolic Control, NIH
In 1968 Krebs and Veech Proposed The Ratio of ATP/ADPxPi is Related to the Both
the Cytosolic and Mitochondrial Redox States Through the GAPDH+ 3PGK Reaction
and Electron Transport
The Energy Level and Metabolic Control in Mitochondria,
Krebs, HA , Veech RL
Pp 329-382, Ed Papa, S. et al, Adriatica Editrice, Bari
The Relation of the Phosphorylation Potential to the
Cytosolic Redox State and The Respiratory Chain was
Proposed in 1968 but It Took 10 and 30 Years to Prove
• In 1968 we did not recognize that changes in free
[Mg2+ ]would alter the value of Keq
• The value of free [Mg2+] in cells was not known and
had to be determined.
• Next Keq had to be determined as function of [Mg2+]
• Finally the value of the mitochondrial membrane
potential had to be determined and related the the
DG of ATP hydrolysis and redox state of the chain.
Free Intracellular [Mg2+] ranges from 0.2 to 1.5 mM and
is Reflected by the [Citrate]/[Isocitrate] Ratio
 [H  ] 

[H  ] 
  Aconi 1 

K ionicAconi1 
K aCit 
K aIsoCit 


2
[Mg ] 


[H  ]  K bMgHIsocit 
[H  ]  K bMgHCit 
  K ionicAconi K bMgCit 

Aconi  K bMgIsocit 



K aIsocit
K aCit




The variation of K’GG-(TPI+LDH) with pH and free [Mg2+].
1
2
pMg
2
3
4
5
6
10 6
Keq
1
10 6
8
7.5
0
7
6.5
6
pH
The Cytosolic Phosphorylation Potential J Biol Chem 1979
VeecH, RL, Cornell, N., Lawson, J., Krebs, HA
The ratio of [PCr]/[Cr} and the ratio of [DHAP]x[Lactate]/[3PG]x[Pyruvate]
give the same ATP/ADPxPi ratio yielding a DG’ of ATP hydrolysis of -53 to
-59 kJ/mol in brain, muscle, liver and red cell .
The creatine kinase and GAPDH + 3PG kinase reastions are in near equilibrium
with the phosphorylation potential.
Free [ADP] is about 20 mmolar with the major of ADP being
segregated within the mitochondrial matrix.
That the free ADP in Cells was Low was Confirmed by NMR
Chance_B_PNAS_1986_83_9458-62_fig4
The Ratio of Free [CoQ]/[CoQH2] Can Be Calculated from
the [fumarate]/[succinate] Ratio
K SuccDH
[fumarate] [CoQH 2 ]

 171.9
[succinate ][CoQ]
And with that the DG ATP between site I
and II
ΔGmito compI -II
2

[succinate
][NAD
]



 nF ( E 'fum/succ  E 'NAD/NADH )  RT ln 
2
 [fumarate ][NADH] 
Bergman C. et al, JPhysChemB 114: 16137, 2010
Sato K. et al, FASEB J 9: 651,, 1995
In Perfused Heart, Ketone Metabolism Increased DG ATP and Mimicked Insulin
E
m/c
Em/c
Mitochondrial area: 32.1 -36.8 %
water: 17 -20 % of ICW
-100
G
GI
GK* GKI*
mV
-110
-120
-130
-140
G
GI* GK* GKI*
H+
NH4+
mV
Eh Q/QH2
20
16
12
8
4
0
-4
-8
4H+
7.6
NH3
NH3
NAD+
4H+
2e-
NADH DH
G
GI* GK* GKI*
G
6.8
GI* GK* GKI*
G
DHB-
4H+
CoQH2-Cyt C
reductase
2e-
ATP4-
ATP4-
ADP3-
3H+
ADP
Succ2Succ CoA
-
Cyt C
Pi
Ox
Cyt aa3
4H+
Red Ox
VO2
-50
-52
FASEB J 9: 651-8, 1995
GI* GK* GKI*
-54
-56
H -Pi
cotransporter
AcAc CoA
2H+
AcAc-
Eh Cyt aa3 Ox/Red
600
580
560
540
GI* GK* GKI*
G
H2PO4H+
H2PO4Cytosolic
[ADP] and [Pi]
H+
G
GI* GK* GKI*
10
8
6
4
*
2
0
G
GI
160
140
120
100
80
*
* 60
40
GK GKI
[ADP]c nmol/ml ICW
1/2 O2
G
Cytosolic
DG of ATP hydrolysis
620
H2O
measured
calculated
GKI
-58
-60
+
2e-
GK
DG ATP hydrolysis
ATP synthase
3H+
TCA cycle KG
Red
4H+
GI
ADP
2-
4H+
Cytochrome
oxidase
G
ATP
ADP3-
ATP
Cit3Fum2-
GI* GK* GKI*
6.6
ATP/ADP
exchanger
NH4+
Pi
mV
kJ/mol
7.2
7.0
Cytosolic pH
7.4
NADH+H
2e-
Succ DH
DG of Q/QH2-NAD /NADH m
mmol/min ICW
7.0
Glut-
+
20
19
18
17
16
15
14
7.6
+
QH2 Q
-50
-52
-54
-56
-58
-60
-62
7.4
7.2
6.8
6.6
H+
-150
Mitochondrial pH
kJ/mol
-260
-270
-280
-290
-300
-310
Mitochondrial
inner membrane
Matrix
[Pi]c mmol/ml ICW
mV
Eh NAD+/NADH m
R. Yasuda, et. al. Cell 1998 93(7):1117-24. “F1-ATPase is a highly
efficient molecular motor that rotates with discrete 120 degree
steps”
Incidence per Year of Disease Phenotype in US
Treatable by Ketones
Substrate Insufficiency
•
•
•
Diabetes types I & II
Alzheimer’s
Heart Failure
Free Radical Toxicity
•
•
Parkinson’s disease
ALS
Hypoxia
• COPD
•
•
MI
Stroke
Insulin Resistance
• Obesity
• Traumatic Head Injury
US Incidence and Source
•
•
•
25 mill
5.1 mill
1.5 mill
NIDDK
Alheimers Org
Am.Heart
•
•
1 mill
0.00035 mill
Parkinson Foundation
ALS Org
•
•
•
30 mill
8.5 mill
6.4 mill
FreeMed
AHA
AHA
•
•
33 mill
1.5 mill
Free Med
CDC
IP Injection of Short Chain Fatty Acids to Starved Rats Increases
CaMgPPi in Liver Mitochondria
• Injecting 2 ml of 2M salts of short chain fatty acids in starved animals
increases vasopressin and increased CaMg PPi to about 4 mmole/g which
was NMR invisible. The increase in CaMgPPi was accompanied by a drop
in the [ATP]/[ADP][Pi] and the DG’ of ATP.
Veech RL et al, AdvExpBiolMed 1986, 194: 617-46
• The metabolism of acetate in rat brain decreases the DG’ of ATP in rat
brain
Pawlosky, R. et al, AlcClinExpRes 2010, 34: 1-7
• The metabolism of D--hydroxybutyrate in rat brain increases the DG’ of
ATP.
Kashiwaya Y. et al, JBiolChem 2010, 285: 25950-6.
Traumatic Brain Injury Afflicates 1.5 million Americans
per Year and Accounts for 20% of Troop Casualties
• Brain Damage in Traumatic brain injury can be
Limited by administration of Cyclosporin which
closes the mitochondrial permiability transition pore.
Buki A. J Neurotrauma 1999, 16: 511-21
Crompton, M. Biochem J. 1999, 341: (pt 2) 233-49
• But cyclosporin A also causes impaired immune
function, limiting its therapeutic use.
Traumatic Brain Injury is Associated with A Low Brain
DG of ATP
• TBI is associated with a decrease in brain O2
consumption, increased brain [Lactate]/[pyruvate]
and increase brain [creatine], all indicative of a
decrease in DG ATP
Casey, PA et al, J Neurotrauma 2008, 25: 603-14
• TBI is associated with a decrease in pyruvate
dehydrogenase activity Sharma, P, J Emerg Trauma
Shock, 2009, 2:67-72
Ketogenic Diets Can Treat TBI and Increase DG
of ATP
• A ketogenic diet limits brain damage after TBI
Prins, ML. J CerebBldFlowMetab 2008 28:1-16
• Metabolism of ketone bodies can by pass the
block in PDH and increase DG of ATP
Sato K. FASEB J 1995 9:651-8
Glucose
Monocarboxyrate
Transporter
(MCT) 1,2,4
Glucose
Glycolysis
PDH
Pyruvate
Lactate
Insulin
Resistance
Acetyl CoA
Acetoacetyl CoA
Succinate
Monocarboxyrate
Transporter
HB 1,2,4
HB
Monocarboxyrate
Transporter
1,2,4
Acetoacetate
NAD
NADH+
Krebs Cycle
Succinyl CoA
Energy
Ketone Body increases
DG ATP
There are no good tools to diagnoses concussion or TBI
• A CAT scan can diagnose intracranial bleeding after
TBI, but give no signal for neuronal injury.
• MRI gives no diagnostic signal in TBI
• MRS shows a decrease in PCr in TBI but is not
applicable to field conditions.
The stopped-flow method and chemical intermediates in enzyme
reactions – a personal essay
Britton Chance
Photosynthesis Research 80: 387–400, 2004.
Fig9
Chance proposed that opening of the mPTP might be visible by NIR providing a diagnotic tool
to diagnoses TBI and evaluate treatment
Fig. 3A. Illustration of a merging of the hemoglobin and water absorption .
spectra to an arbitrary scale to show their spectral overlap and the optimal
“windows for minimal crosstalk of the three metrics to be studied.
Fig. 3B. Sensitivity of water absorption spectrum to temperature. Thermal
difference spectra of water in a 1 mm path length, baseline 37o with
lowering to 17 o and rising to 50 o, showing that 970 nm is an appropriate
measuring wavelength and 1010 nm is a suitable reference wavelength. A
signal increment of 0.010 optical density per degree for a 1 cm optical path
is calculated
The stopped-flow method and chemical intermediates in enzyme
reactions – a personal essay
Britton Chance
Photosynthesis Research 80: 387–400, 2004.
Fig9