Enzyme Assay

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http//fiehnlab.ucdavis.edu/teaching/ folder.20
07-08-20.0671728135/
Fri Nov 02 Assay of enzyme activities
reading list Mo Nov 05 Mass
spectrometry fundamentals Wed Nov 07
Mass spectrometry quantification and
identification Fri Nov 09 Primary
metabolism overview and integration Mo Nov
12 Veteran's Day Wed Nov 14 Homework
discussion I Fri Nov 16 Animal models
for studying metabolic networks Mo Nov 19
Regulation of glycogen breakdown Wed Nov
21 Inborn errors of glycogen metabolism Fri
Nov 23 Thanksgiving Mo Nov 26
Metabolic networks in humans from KO to SNP
variants Wed Nov 28 Homework discussion
II Fri Nov 30 Flux analysis,
stoichiometry and elementary modes Mo Dec
03 (Bio)chemical databases (Guest lecturer Dr.
Tobias Kind) Wed Dec 05 Tools for
modeling metabolism (Guest lecturer Dr. Tobias
Kind) Fri Dec 07 Homework discussion III
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Enzyme Assays
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(1) Development of an assay

• A useful enzyme assay must meet four criteria
• (a) absolute specificity
• (b) high sensitivity
• (c) high precision accuracy
• (d) convenience

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(A) Absolute specificity

• Most enzyme assays monitor disappearance of a
  substrate or appearance of a product
• Ensure that only one enzyme activity is
  contributing to the monitored effect!

Ensure absence of PEPcarboxylasePEP HCO3- ?
OAA Pi absence of pyruvate kinasePEP ADP ?
pyruvate ATP absence of PEPcarboxytransphosphory
lasePEP CO2 Pi ? OAA PPi
e.g. PEPCKPEP CO2 GDP ? OAA GTP
Study cofactor requirements and product
identification under a variety of conditions /
scientific papers. Examples as above are found
for almost any enzyme. Be aware of possible
reactions that may contribute to a given product
accumulation or substrate utilization!
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(B) High sensitivity

• e.g. for purification, specific activities of
  most enzymes are very low.Therefore, the assay
  must be highly sensitive.

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(C) High precision

• The accuracy and precision of an enzyme assay
  usually depend on the underlying chemical basis
  of techniques that are used.
• For example, if an assay is carried out in buffer
  of the wrong pH, the observed rates will not
  accurately reflect the rate of enzymatically
  produced products

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Six major characteristics of a protein solution

• Six major characteristics of a protein solution
  warrant consideration
• pH
• Degree of oxidation
• Heavy metal contamination
• Medium polarity
• Protease contamination
• temperature

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pH

• pH values yielding the highest reaction rates are
  not always those at which the enzyme is most
  stable. It is advisable to determine the pH
  optima for enzyme assay and stability separately.
• For protein purifications Buffer must have an
  appropriate pKa and not adversely affect the
  protein(s) of interest. Buffer capacity may be
  higher for tissues with large vacuoles such as
  plants and fungi.

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Degree of oxidation

• Most proteins contain free SH groups. One or more
  of these groups may participate in substrate
  binding and therefore are quite reactive.
• Upon oxidation, SH turn form intra- or
  inter-molecular S-S bonds, which usually result
  in loss of enzyme activity.
• A wide variety of compounds are available to
  prevent disulfide bond formation
  2-mercaptoethanol, cysteine, reduced glutathione,
  and thioglycolate. These compounds are added to
  protein solutions at concentration ranging from
  10-4 to 5 ?10-3 M (excess because equilibrium are
  near unity). Dithiothreitol is advantageous
  (lower amounts needed) because of formation of
  stable six-ring.
• Antioxidants against quinones (e.g. protein
  isolation from plants) by polyvinylpyrrolidone.

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Heavy metal contamination

• SH groups may react with heavy metal ions such as
  Pb, Fe, Cu stemming from buffers, ion exchange
  resins or even the water in which solutions are
  prepared.
• If trace amounts of heavy metals continue to be a
  problem, EDTA (ethylenediaminetetraacetic acid)
  may be included in the buffer solutions at a
  concentration of 1 to 3 ?10-4M. The compounds
  chelates most, if not all, deleterious metal ions.

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Protease or nuclease contamination

• During cell breakage, proteases and nucleases are
  liberated.
• PMSF (phenylmethylsulfonyl fluoride)
• a commonly used protease inhibitor

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Temperature

• Not all proteins are most stable at 0 C, e.g.
  Pyruvate carboxylase is cold sensitive and may be
  stabilized only at 25 C.
• Freezing and thawing of some protein solutions is
  quite harmful. If this is observed, addition of
  glycerol or small amounts of dimethyl sulfoxide
  to the preparation before freezing may be of
  help.
• Storage conditions must be determined by trial
  and error for each protein.

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More on keeping proteins for enzyme assays

• Proteins requiring a more hydrophobic environment
  may be successfully maintained in solutions whose
  polarity has been decreased using sucrose,
  glycerol, and in more drastic cases, dimethyl
  sulfoxide or dimethylformamide. Appropriate
  concentrations must usually be determined by
  trial and error but concentrations of 1 to 10
  (v/v) are not uncommon.
• A few proteins, on the other hand, require a
  polar medium with high ionic strength to maintain
  full activity. For these infrequent occasions,
  KCl, NaCl, NH4Cl, or (NH4)2SO4 may be used to
  raise the ionic strength of the solution.

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Protein purification for testing novel enzymes
series of isolation and concentration procedures

• Major techniques for the isolation and
  concentration of proteins differential
  solubility, ion exchange chromatography,
  absorption chromatography, molecular sieve
  techniques, affinity chromatography,
  electrophoresis.
• Which technique will be successful? .trial and
  error.

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Coupled enzyme assays
Most enzyme assays monitor disappearance of a
substrate or appearance of a product So, how to
measure?

• If neither the substrates nor products of an
  enzyme-catalyzed reaction absorb light at an
  appropriate wavelength,the enzyme can be assayed
  by linking to another enzyme-catalyzed reaction
  that does involve a change in absorbance.
• The second enzyme must be in excess,so that the
  rate-limiting step in the linked assay is the
  action of the first enzyme.

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Coupled enzyme assays

• Most useful, most frequent
• Not at all foolproof!

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Errors and artifacts in coupled enzyme assays
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A little reminder on Glycolysis stage 1
phosphofructokinase activates for C-C cleavage
Mg2
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A little reminder on Glycolysis DGo and DG in
heart muscle
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A little reminder on Glycolysis Allosteric sites
in PFK
In (mammals), Phosphofructokinase (PFK) is a 340
kd tetramer, which enables it to respond
allosterically to changes in the concentrations
of the substrates fructose 6-phosphate and ATP
In addition to the substrate-binding sites,
there are multiple regulatory sites on the
enzyme, including additional binding sites for
ATP
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A little reminder on Glycolysis/Gluconeogenesis
High ATP levels inhibit PFK activity
High ATP levels will change the kinetics of PFK
from an asymptotic curve to a sigmoidal one
The sigmoidal curve reflects the reduced need
for glycolysis at high energy levels in the cell
This base ATP-dependent curve of PFK can then be
further modulated by the concentration of
fructose 2,6-bisphosphate
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A little reminder on glycolysis .and
gluconeogenesis
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Fructose 2,6-Bisphosphate is an Activator of
PFK Fructose 2,6-bisphosphate (F-2,6-BP) is a
second allosteric effector of PFK It functions
as an activator that overrides the inhibitory
effect of ATP
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F-2,6-BP Levels are Controlled by a Bifunctional
Enzyme                                          
                                                  
                                                  
                                                  
                                                  
                                                  
                                                  
                                                  
                                                  
                                                  
                  The concentration of
Fructose 2,6-Bisphosphate (F-2,6-BP) in cells is
determined by a bifunctional enzyme,
phosphofructokinase 2 / fructose bisphosphatase2
((PFK2/FBPase2), to provide an additional level
of control for PFK activity F2,6-BP is formed by
phosphorylation of fructose 6-phosphate in a
reaction catalyzed by PFK2 The resulting
phosphoryl group on the C-2 can then be removed
by the phosphatase FBPase2
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Reminder of gluconeogenesis by glucagon/cAMP
cascade plus allosteric activation of PFK by
Fructose-2,6-bisphosphate
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cited 19x F6P may contain 0.001 F2,6BP
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imidodiphosphate is contaminated by 2 PPi and
is actually inhibiting PFP.
ATP was contaminated by 0.3 PPi, and PPi is an
activator of PFK
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(No Transcript)
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Errors and artifacts in coupled enzyme assays
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Errors and artifacts in coupled enzyme assays

• Strategy
• Optimize your assay.(1) pH (2) substrate
  concentrations should not be too large (3) conc.
  of coupled enzymes should be not too large (4)
  vary buffers and counter ions. Compromise
  between your enzyme and the requirements for
  the coupled enzymes. (5) Consider isozymes.
• Consider particularities of your enzyme and
  coupled enzymes.
• Question anomalous response in changing E or
  unusual kinetics (bursts, lag times)
• Use substrates from different vendors
• Check that reaction does not stop before
  depletion of limiting substrate/cofactor

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If one coupled enzyme assay is difficult to
control 23 assays must be easy !?
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Robotized multi-enzyme assay

• Measurement of enzymome not possible
• Group subsets of enzymes in modules that share
  common detection method.
• Cycling assays used. (pseudo zero order, rate
  depending on metabolite
• In combination with stopped assay, some 104fold
  more sensitive.

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Cycling assay?
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Dye- or fluorescent labels
Classic substrates
Novel substrates
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Real-time labels
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In vivo assay FRET(fluorsc. resonance energy
transfer)
Wolf Frommer Carnegie
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HepG2 cells expressing glucose-sensitive FRET
nanosensor in the cytosol. Addition of 5 mM
glucose
Red color indicates low internal glucose levels,
green color shows high internal glucose
concentrations. Ratio red/green over time.
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Further reading