http://fiehnlab.ucdavis.edu/teaching/folder.2007-08-20.0671728135/

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

Enzyme Assays

(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

(A) Absolute specificityMost enzyme assays monitor disappearance of a substrate or

appearance of a product

Ensure that only one enzyme activity is contributing to the

monitored effect!

e.g. PEPCK

PEP + CO2 + GDP ⇋ OAA + GTP

Ensure

absence of PEPcarboxylase

PEP + HCO3- → OAA + Pi

absence of pyruvate kinase

PEP + ADP → pyruvate + ATP

absence of PEPcarboxytransphosphorylase

PEP + CO2 + Pi ⇋ OAA + PPi

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!

(B) High sensitivity

e.g. for purification, specific activities of most enzymes are very low.

Therefore, the assay must be highly sensitive.

(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

Six major characteristics of a

protein solutionSix major characteristics of a protein solution warrant

consideration

1. pH

2. Degree of oxidation

3. Heavy metal contamination

4. Medium polarity

5. Protease contamination

6. temperature

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.

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 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.

Protease or nuclease

contaminationDuring cell breakage, proteases and nucleases are

liberated.

PMSF (phenylmethylsulfonyl fluoride):

a commonly used protease inhibitor

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.

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 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.

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.

Most enzyme assays monitor disappearance of a substrate or

appearance of a product…

So, how to measure?

Coupled enzyme assays• Most useful, most frequent

• Not at all foolproof!

Errors and artifacts

in coupled enzyme assays

Mg2+

A little reminder on Glycolysis

stage 1: phosphofructokinase activates for C-C cleavage

A little reminder on GlycolysisGo` and G in heart muscle

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

A little reminder on Glycolysis

Allosteric sites in PFK

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

A little reminder on Glycolysis/Gluconeogenesis

High ATP levels inhibit PFK activity

A little reminder on glycolysis

….and gluconeogenesis

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:

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

Reminder of gluconeogenesis by glucagon/cAMP cascade

plus allosteric activation of PFK by Fructose-2,6-bisphosphate

cited 557xcited 157x

cited 82x

cited 19x

“F6P may contain

~ 0.001% F2,6BP”

…ATP was contaminated by 0.3% PPi,

and PPi is an activator of PFK…

PFP has

…imidodiphosphate is

contaminated by 2% PPi and is

actually inhibiting PFP.

…auxiliary enzymes were contaminated with

UDP pyrophosphorylase…

…auxiliary enzymes were contaminated with

adenylate kinase…

Errors and

artifacts

in coupled

enzyme assays

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 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 10^4fold more sensitive.

Cycling assay?

Dye- or fluorescent labels

Classic substrates Novel substrates

Real-time labels

In vivo assay FRET(fluorsc. resonance energy transfer)

Wolf Frommer

Carnegie

Red color indicates low internal glucose levels, green color shows high internal glucose

concentrations. Ratio red/green over time.

HepG2 cells expressing glucose-sensitive FRET

nanosensor in the cytosol. Addition of 5 mM glucose

Further reading