http://fiehnlab.ucdavis.edu/teaching/ folder.2007-08-20.0671728135/Fri Nov 02 Assay of enzyme activities reading listMo Nov 05 Mass spectrometry: fundamentalsWed Nov 07 Mass spectrometry: quantification and identificationFri Nov 09 Primary metabolism: overview and integrationMo Nov 12 Veterans DayWed Nov 14 Homework discussion IFri Nov 16 Animal models for studying metabolic networksMo Nov 19 Regulation of glycogen breakdownWed Nov 21 Inborn errors of glycogen metabolismFri Nov 23 ThanksgivingMo Nov 26 Metabolic networks in humans: from KO to SNP variantsWed Nov 28 Homework discussion IIFri Nov 30 Flux analysis, stoichiometry and elementary modesMo 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
(1) Development of an assayA useful enzyme assay must meet four criteria:(a) absolute specificity(b) high sensitivity(c) high precision & accuracy(d) convenience
(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 PEPcarboxylase e.g. PEPCK PEP + HCO3- → OAA + Pi absence of pyruvate kinase PEP + CO2 + GDP ⇋ OAA + GTP PEP + ADP → pyruvate + ATP absence of PEPcarboxytransphosphorylase PEP + CO2 + Pi ⇋ OAA + PPiStudy 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!
(B) High sensitivitye.g. for purification, specific activities of most enzymes are very low. Therefore, the assay must be highly sensitive.
(C) High precisionThe 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
Six major characteristics of a protein solutionSix major characteristics of a protein solution warrant consideration4. pH5. Degree of oxidation6. Heavy metal contamination7. Medium polarity8. Protease contamination9. temperature
pHpH 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.
Degree of oxidationMost 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.
Heavy metal contaminationSH 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.
Protease or nuclease contaminationDuring cell breakage, proteases and nucleases are liberated.PMSF (phenylmethylsulfonyl fluoride):a commonly used protease inhibitor
TemperatureNot 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.
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.
Protein purification for testing novel enzymes: series of isolation and concentration proceduresMajor 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.
Most enzyme assays monitor disappearance of a substrate or appearance of a product…So, how to measure? Coupled enzyme assays • 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.
Coupled enzyme assays• Most useful, most frequent• Not at all foolproof!
A little reminder on Glycolysisstage 1: phosphofructokinase activates for C-C cleavage Mg2+
A little reminder on Glycolysis ∆Go` and ∆G in heart muscle
A little reminder on Glycolysis Allosteric sites in PFKIn (mammals), Phosphofructokinase (PFK) is a 340 kd tetramer, which enables it to respondallosterically to changes in the concentrations of the substrates fructose 6-phosphate and ATPIn addition to the substrate-binding sites, there are multiple regulatory sites on the enzyme, includingadditional binding sites for ATP
A little reminder on Glycolysis/Gluconeogenesis High ATP levels inhibit PFK activityHigh ATP levels will change the kinetics of PFK from an asymptotic curve to asigmoidal one:The sigmoidal curve reflects the reduced need for glycolysis at high energy levelsin the cellThis base ATP-dependent curve of PFK can then be further modulated by theconcentration of fructose 2,6-bisphosphate
A little reminder on glycolysis ….and gluconeogenesis
Fructose 2,6-Bisphosphate is an Activator of PFKFructose 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:
F-2,6-BP Levels are Controlled by a Bifunctional EnzymeThe concentration of Fructose 2,6-Bisphosphate (F-2,6-BP) in cells is determined by abifunctional enzyme, phosphofructokinase 2 / fructose bisphosphatase2 ((PFK2/FBPase2),to provide an additional level of control for PFK activityF2,6-BP is formed by phosphorylation of fructose 6-phosphate in a reaction catalyzed byPFK2The resulting phosphoryl group on the C-2 can then be removed by the phosphataseFBPase2
Reminder of gluconeogenesis by glucagon/cAMP cascadeplus allosteric activation of PFK by Fructose-2,6-bisphosphate
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
If one coupled enzyme assay is difficult to control… …23 assays must be easy !?
Robotized multi-enzyme assayMeasurement 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 10^4fold more sensitive.
In vivo assay FRET (fluorsc. resonance energy transfer)Wolf FrommerCarnegie
HepG2 cells expressing glucose-sensitive FRET nanosensor in the cytosol. Addition of 5 mM glucoseRed color indicates low internal glucose levels, green color shows high internal glucoseconcentrations. Ratio red/green over time.