Towards a Kinetic Model of the Hepatocyte Core Metabolism by slappypappy118

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									Towards a Kinetic Model of the
 Hepatocyte Core Metabolism

Matthias König
Charite Berlin

SABIO-RK User Meeting
Heidelberg [15/06/09]
                  Long term objectives
●   Kinetic model of core hepatocyte
    –   energy metabolism
    –   carbohydrate metabolism
    –   amino acid and nucleotide metabolism
    –   fixation / detoxification NH3
●   Functionality
    –   Constant blood glucose level
    –   NH3 fixation and detoxification
    –   Keto body synthesis
    –   ...
Workflow for kinetic
modeling
                           Overview
●   [1] Network reconstruction
        –   HepatoSys
        –   HepatoCore
●   [2] SABIO-RK
        –   Collection of kinetic data
        –   Web-services
●   [3] Towards a kinetic model
        –   Information integration
        –   Example rate equations
    Network reconstruction in HepatoSys
●   metabolic network reconstruction of human
    hepatocytes
●   manually curated and evidence based
●   tracking type and strength of evidence with
    consistent use of ontologies
●   development of tools for network
    reconstruction, validation, storage, data
    query and retrieval
●   FBA and kinetic simulation
  HepatoCore for kinetic modeling



Pathways
● glycolysis / gluconeogenesis

● pentose phosphate pathway
                                   Subnetwork of HepatoNet
                                   ● ~ 300 reactions
● citrate cycle
                                   ● ~ 100 transporters
● amino acid metabolism

● purine / pyrimidine metabolism

● keto body synthesis
                                   Validation
                                   ● Structural methods
● folate metabolism
                                   ● Functionality tests

                                   ● FBA simulations
Compartments
● cytosol

● mitochondrion

● nucleus

● er

● blood
[2] SABIO-RK for Kinetic Modeling
          Web Services with Python




from SOAPpy import WSDL

# Get WSDL file and create server connection
wsdl = 'http://sabio.bioquant.uni-heidelberg.de/sabiows/sabiork.jws?wsdl'
# Create server connection
server = WSDL.Proxy(wsdl)

# retrieve information from SABIO-RK
reaction_id = server.getReactionIDByKEGG(rid)
print server.getReactionInstanceIds(reaction_id)
SABIO-RK Web Services
                                                    Km values glycolysis
                                                    Km Values Glycolysis

             Nucleoside-diphosphate kinase

                           Pyruvate kinase

                     6-Phosphofructokinase

            Glucose-6-phosphate isomerase

           Polyphosphate phosphohydrolase

                      Phosphoglucomutase

                   Phosphoglycerate kinase

            Fructose-bisphosphate aldolase

Glyceraldehyde-3-phosphate dehydrogenase

                               Hexokinase

                               Glucokinase

                Phosphopyruvate hydratase

UTP-glucose-1-phosphate uridylyltransferase

                  Phosphoglycerate mutase

               Bisphosphoglycerate mutase

                    Glucose-6-phosphatase

                Sugar-terminal-phosphatase

                  Fructose-bisphosphatase

                          Acylphosphatase

               Triose-phosphate isomerase

                                              0      20      40       60       80      100      120   140   160   180   200

                                              Homo Sapiens   Vertebrata    Eukaryota   Prokaryota
                                 Kinetic Laws Glycolysis                                                                                        Km Values Glycolysis

             Nucleoside-diphosphate kinase                                                                                   Nucleoside-diphosphate kinase
                            Pyruvate kinase                                                                                                Pyruvate kinase
                      6-Phosphofructokinase                                                                                         6-Phosphofructokinase
            Glucose-6-phosphate isomerase                                                                                   Glucose-6-phosphate isomerase
           Polyphosphate phosphohydrolase                                                                                  Polyphosphate phosphohydrolase
                       Phosphoglucomutase                                                                                             Phosphoglucomutase
                    Phosphoglycerate kinase                                                                                        Phosphoglycerate kinase
            Fructose-bisphosphate aldolase                                                                                  Fructose-bisphosphate aldolase
Glyceraldehyde-3-phosphate dehydrogenase                                                                         Glyceraldehyde-3-phosphate dehydrogenase
                                Hexokinase                                                                                                     Hexokinase
                               Glucokinase                                                                                                    Glucokinase
                Phosphopyruvate hydratase                                                                                      Phosphopyruvate hydratase
UTP-glucose-1-phosphate uridylyltransferase                                                                     UTP-glucose-1-phosphate uridylyltransferase
                   Phosphoglycerate mutase                                                                                       Phosphoglycerate mutase
               Bisphosphoglycerate mutase                                                                                       Bisphosphoglycerate mutase
                     Glucose-6-phosphatase                                                                                          Glucose-6-phosphatase
                Sugar-terminal-phosphatase                                                                                      Sugar-terminal-phosphatase
                   Fructose-bisphosphatase                                                                                         Fructose-bisphosphatase
                           Acylphosphatase                                                                                                Acylphosphatase
                Triose-phosphate isomerase                                                                                      Triose-phosphate isomerase

                                                     0          10          20      30        40         50     60                                                   0       100      200         300    400    500     600
                                                                                                                                                 Specific Activity Glycolysis
                                                                                                                                        Homo Sapiens Vertebrata Eukaryota Prokaryota
                                 Kcat Values Glycolysis

               Nucleoside-diphosphate kinase                                                                                  Nucleoside-diphosphate kinase
                              Pyruvate kinase                                                                                                Pyruvate kinase
                        6-Phosphofructokinase                                                                                         6-Phosphofructokinase
              Glucose-6-phosphate isomerase                                                                                  Glucose-6-phosphate isomerase
             Polyphosphate phosphohydrolase                                                                                 Polyphosphate phosphohydrolase
                         Phosphoglucomutase                                                                                            Phosphoglucomutase
                      Phosphoglycerate kinase                                                                                       Phosphoglycerate kinase
              Fructose-bisphosphate aldolase                                                                                 Fructose-bisphosphate aldolase
  Glyceraldehyde-3-phosphate dehydrogenase                                                                       Glyceraldehyde-3-phosphate dehydrogenase
                                  Hexokinase                                                                                                      Hexokinase
                                 Glucokinase                                                                                                     Glucokinase
                  Phosphopyruvate hydratase                                                                                      Phosphopyruvate hydratase
  UTP-glucose-1-phosphate uridylyltransferase                                                                    UTP-glucose-1-phosphate uridylyltransferase
                     Phosphoglycerate mutase                                                                                       Phosphoglycerate mutase
                 Bisphosphoglycerate mutase                                                                                     Bisphosphoglycerate mutase
                       Glucose-6-phosphatase                                                                                         Glucose-6-phosphatase
                  Sugar-terminal-phosphatase
                                                                                                                                 Sugar-terminal-phosphatase
                     Fructose-bisphosphatase
                                                                                                                                   Fructose-bisphosphatase
                             Acylphosphatase
                                                                                                                                           Acylphosphatase
                  Triose-phosphate isomerase
                                                                                                                                Triose-phosphate isomerase
                                                         0       20         40     60    80        100    120
                                                                                                                                                                     0         20        40         60     80     100         120
                         Homo Sapiens   Vertebrata       Eukaryota    Prokaryota
                                                                                                                                         Homo Sapiens   Vertebrata       Eukaryota   Prokaryota
[3] Towards kinetic model
Example 1: Glucose Transport
                                                        2V8N – Wildtype structure of lactose permease




GLUT2 1 D-Glucose [ext] <-> 1 D-Glucose [cytosol]
Glucose is a polar molecule, it does not readily diffuse across the plasma membrane.
[Olson1996]
… facilitative glucose transporters are characterized by a high degree of stereoselectivity,
providing for the bidirectional transport of glucose, with passive diffusion solely down its
concentration gradient.
GLUT2 protein is found on the basolateral membrane surfaces of liver cells, pancreatic beta-cells,
small intestine and kidney, where its relatively high Km results in transport activity in direct
proportion to the physiological range of glucose concentrations.

As the entire antiport process must occur at a physiologically realistic rate and be responsive to
changes in substrate concentrations on both sides of the membrane, reasonable values for the rate
constants are needed for the individual stages of the reaction cycle. Like other antiporters, GlpT
obeys Michaelis-Menten kinetics and operates via a ping-pong mechanism. [Law2008]

Glucose uptake studies performed in liver (127) or isolated hepatocytes demonstrated that:
 ●   The Km for glucose was relatively high (~15-20mM (26))
 ●   The transport process was symmetrical, i.e. the Km was the same (~20mM) for glucose
     influx or efflux in zero-trans or in equilibrium exchange conditions (25,26)
 ●   GLUT2 was functionally expressed in in bacteria (116) and in Xenopus oocytes after injection of
     in vitro synthesized mRNA. In Xenopus oocytes the Km for 3-O-methylglucose was 42mM in
     equilibrium exchange conditions (41) and 16.2mM for 2-doxy-D-glucose uptake (13). In
     transfected AtT20-cells, the Km for uptake by GLUT2 was ~17mM.
                                GLUT2 kinetics
●   GLUT2 is the predominant facilitated diffusion glucose transporter in hepatocytes
●   low-affinity glucose transporter with a high turnover rate
●   symmetrical transport
●   Michaelis Menten Kinetics



Reversible Michaelis Menten
Km = 20 mM
Keq = 1

               Vmax              [ glccyto ]
                    [glc ext ]−             
                Km                  K eq
          v 1=
                   [glc ext ] [ glc cyto ]
                1           
                      Km           Km
Example 2: Glucokinase
                               Glucokinase
●   wild-type and mutant enzymes show cooperative/sigmoidal glucose-dependent
    kinetics [Pubmed: 660810]
●
    GK differs in its kinetic properties from the other hexokinase isoforms in having a low
    affinity for glucose and sigmoidal kinetics
●    ...This suggests that the positive cooperativity of monomeric glucokinase obeys
    the "mnemonical mechanism" rather than the well-known concerted model.
    These structures also revealed an allosteric site through which small molecules may
    modulate the kinetic properties of the enzyme.
●
    In the liver, GK activity is regulated by hormonal control of gene expression
    and by a 68-kDa GK regulatory protein (GKRP), which inhibits GK competitively
    with respect to glucose
●
    Glucokinase of the beta-cell is induced or activated by glucose in contrast to
    liver glucokinase, which is regulated by insulin. Tissue-specific regulation
    corresponds with observations that liver and pancreatic beta-cell glucokinase are
    structurally distinct.
        SABIO-RK data for glucokinase
●   Multiple entries in SabioRK for the human glucokinase protein
    P35557
●   But only two wildtype entries for liver, Homo sapiens and D-glucose
    and ATP as substrates

Sabio: 2580                                 Sabio: 2589
 ● variation of glucose (constant [ATP])     ● variation of ATP (constant [glc])

 ● Hill kinetics                             ● Michaelis Menten kinetics

         vmax                 12.3 mU/ml;            vmax              12.3 mU/ml;
         Km[glc]              5.7 mM                 Km[ATP]:          0.26 mM
         [ATP]                1    mM;               [glc]             100 mM;
         [E]                  60 nM                  [E]               60 nM
         n                    1.6


                              n
                   v max [glc ]                            v max [ ATP]
        v=         glc n          n
                                                    v=      ATP
              K  [ glc]
                   m
                                                         K m [ ATP]
                   n
        v max [glc ]
v=
      K glc n [ glc]n
         m




        v max [ ATP]
v=      ATP
      K m [ ATP]
                                                                 n
                                                      v max [glc ]
                                               v=      glc n          n
                                                     K m  [ glc]


                                                       v max [ ATP]
                                               v=      ATP
                                                     K m [ ATP]




               [ ATP]        [glc ]n
v =v max   ATP            n      glc n
                                           
            K m [ ATP] [glc ]  K m 

=1.11
               [ ATP]        [glc ]n
v =v max   ATP            n      glc n
                                           
            K m [ ATP] [glc ]  K m 
                     Work in progress
●   Further development of rate equations
●   Simulations of glycolysis with HepG2 flux
    distributions started
●   Capture the main regulations in the equations
●   Information integration and database
    implementation
●   Methods to combine multiple reaction
    measurements in one rate equation (combine
    the slices)
                               Thanks
●   Hermann-Georg Holzhütter
●   Sascha Bulik
●   Christian Bölling
●   Christoph Gille
●   Sabrina Hoffmann
●   Andreas Hoppe
●   Michael Weidlich

                                     HepatoNet Contributors
                                 ●   C. Bölling, C. Gille, S. Hoffmann, A.
                                     Hoppe, S. Bulik, K. Hübner, H. G.
                                     Holzhütter & al.
                          Sabio Ideas
●   Webinterface not good at all
        –   Warenkorb (captured entries of interest)!!! would help a l
        –   Search should have filter options (otherwise can start
              always from scratch)
        –   Some kind of ontology for the terms ??? especially for
              species,
        –   Export format in webinterface
        –   Get entries for protein !!!
        –   Combined kinetics ???
        –   Measurements from the same paper.
        –   Your session expired !!! In most cases lots of time
              between looking at the next entry → have to start from
              scratch

								
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