Enzyme Inhibition: AimsReview of Practicals • To demonstrate the digestions of caesin by protease enzymes BIOC2101 • To provide an understanding of how an enzyme may be inhibited initially as a result of synthesis as a zymogen Enzyme Inhibition: Aims • To provide an understanding of how the zymogen precursors of protease enzymes are activated in the small intestine Background • Parts A, B and C all use an assay for protease activity • The substrate is casein • The enzyme is a pancreatic protease • To illustrate the specific inhibition of proteases and the role of pancreatic trypsin inhibitors Background • The protease hydrolyses internal bonds within the casein to produce smaller peptides • This reaction can be stopped by adding TCA, which denatures and precipitates the proteins (enzyme and large casein molecules) • Centrifugation ensure only the small peptides remain in the supernatant Background • Bradford's reagent is used to measure the amount of peptides present in the supernatant • The reagent produces an intense blue colour with all peptides and proteins (measured at 620 nm) 1 Part A Part A Substrate and buffer RO water Trypsin inhibitor Trypsin Trypsin 0 min 10 min 20 min 30 min • Trypsin inhibitor acts to inhibit the protease activity of trypsin 0 min 10 min 20 min 30 min Reactions stopped Bradford assay performed Part B Substrate and buffer RO water Trypsin inhibitor chymotrypsin chymotrypsin 0 min 10 min 20 min 30 min Part B • Trypsin inhibitor does not inhibit the protease activity of chymotrypsin 0 min 10 min 20 min 30 min Reactions stopped Bradford assay performed Part C • Chymotrypsinogen is a zymogen • Chymotrypsinogen is activated by trypsin Part C chymotrypsinogen trypsin trypsin 0 min 5 min 10 min inhibitor 15 min 20 min 35 min Casein added Reactions stopped Bradford assay performed 2 . 6-bisP NAD+ + Pi NADH + H+ 2 x 1.Assay system for lactate" " Expt A investigated what cofactors are necessary for glycolysis (fructose-1." " .6-bisP DHAP 2 x PEP ADP " NADH NAD+ ATP 2 x PYR Typical Results" " Addition ! !ΔA" Water " "0.Glycolysis(Prac.3-bisPG ADP 2 x 2-PG DHAP Experiment A ADP G-6-P ATP 2 x PYR " NADH NAD+ lactate " 3 .cal( Part C • The longer the incubation. e.10" NAD+/Pi " "0.! ADP --> AMP + Pi! (and phosphate release from AMP and NAD+)" GLUCOSE ATP lactate ADP G-6-P " " lactate " Experiment A F-6-P ATP Why might there be some glycolysis in the absence of NAD+?! GAP NAD+ + Pi ! NADH + H+ NAD+ binds very 2 x 1.45" NAD+/ADP" "0.6-bisP DHAP ADP F-1.Dialysed rat skeletal muscle extract.04" "" NADH NAD+ ATP 2 x PYR " Experiment A ADP G-6-P GAP GAP NADH + H+ 2 x 1.3-bisPG ADP tightly to some ATP enzymes and is 2 x 3-PG difficult to remove 2 x 2-PG completely by dialysis! H2O ADP F-1.6-bisP GAP Expect to find that ADP.3-bisPG ADP ATP 2 x 3-PG 2 x 2-PG H2O 2 x PEP ADP Why was there some glycolysis in the absence of Pi?! ! Phosphatases.02" ADP/Pi " "0. the more chymotrypsinogen is activated • The trypsin inhibitor stopped the zymogen from being activated and prevents trypsin from acting on the casein • The more chymotrypsin is present.6-bisP --> lactate)" " Expt B investigated the rate limiting step in glycolysis (glycolytic intermediate --> lactate)" GLUCOSE ATP Experiment A ADP G-6-P F-6-P ATP F-6-P ATP ADP F-1. the more casein will be digested GLUCOSE ATP Provided with:-" .02" NAD+/ADP/Pi "0. containing all the enzymes for glycolysis and other soluble cytoplasmic enzymes. but no mitochondrial enzymes and no metabolites/cofactors.3-bisPG ADP ATP 2 x 3-PG DHAP NAD+ + Pi ATP 2 x 3-PG 2 x 2-PG H2O 2 x PEP ADP NADH ATP 2 x PYR H2O 2 x PEP ADP " NAD+ lactate GLUCOSE ATP F-6-P ATP ADP F-1.g. Pi and NAD+ are all required for rapid glycolysis" NAD+ + Pi NADH + H+ 2 x 1. 6-bisP" GAP" 1.ons(during(first(glycolysis(and(then(the(lactate( assays?( Glycolysis" NADH + H+" DHAP Why was NO lactate formed starting from 1.ons(in(the( opposite(direc.6-bisP ATP 2 x PYR " lactate" GLUCOSE ATP F-6-P ATP 2 x PEP ADP Typical Results" " Addition !ΔA" Water "0.on(Techniques( 3 types of chromatography:-" " Partition (thin layer)" " Permeation (gel filtration)" " Ion exchange" " All involve the differential distribution of molecules between a stationary phase and a mobile phase" 4 .3-bisPG ADP ATP 2 x 3-PG 2 x 2-PG H2O 2 x PEP ADP NADH ATP 2 x PYR DHAP GAP 2 x 2-PG H2O 2 x PEP ADP " NAD+ ADP F-1.08" F-6-P" "0.02" Glucose "0.6-bisP ADP F-1.15" F-1.3-bisPG" + all necessary cofactors" + NAD + Pi NADH + H+ 2 x 1.3-bisPG ADP lactate" GLUCOSE ATP Experiment B ADP G-6-P Relatively low pH of 7.3-bisPG "0.4 (high [H+]) lactate" favours lactate formation" NAD+" Lactate assay" Relatively high pH of 9.GLUCOSE ATP GLUCOSE ATP Experiment B ADP G-6-P F-6-P ATP F-6-P ATP ADP F-1.3bisPG?! GAP NAD+ + Pi ! X!NADH + H+ NO NADH was 2 x 1.4 (low [H+]) NAD+" NADH + H+" lactate" pyruvate" favours pyruvate formation" reacts non-enzymically with hydrazine" ATP 2 x PYR lactate " Separa.6-bisP Why was lactate formed starting from glucose and F-6-P GAP NAD+ + Pi when no ATP was NADH + H+ added?! 2 x 1.50" 1.6-bisP "0.3-bisPG ADP ! ATP Adenylate kinase!!! 2 x 3-PG 2ADP <--> AMP + ATP! 2 x 2-PG H2O NADH " pyruvate" 2 x 2-PG " NADH NAD+ " How(can(lactate(dehydrogenase(catalyse(reac.45" GAP " "0.6-bisP DHAP GAP Start with:-" Glucose" F-6-P" F-1.02" NADH NAD+ ATP 2 x PYR F-6-P ATP DHAP NAD+ + Pi ATP 2 x 3-PG Experiment B ADP G-6-P rate limiting step" NADH + H+ 2 x 1.3-bisPG ADP produced to reduce ATP pyruvate to lactate!!! 2 x 3-PG H2O 2 x PEP ADP NAD+ lactate Experiment B ADP G-6-P ADP F-1. g. Molecules with no net charge (or with a net charge the same as that on the ion exchange column) will pass through with the mobile phase. but not to large molecules. that is accessible to relatively small molecules." " More polar molecules partition into the stationary aqueous phase and do not migrate far from the origin.( Yellow ferricyanide forms a diffuse slow moving band! Red/brown hemoglobin forms a much tighter fast moving band! Why(does(the(Rf(change(in(different( solvents?( Different solvents may be more or less polar." " e. leucine)" What(is(the(func." -OOC-CH + 2-CH2-CH(NH3 )COO " HOOC-CH2-CH2-CH(NH3+)COO-" HOOC-CH2-CH2-CH(NH3+)COOH" Gel(filtra.g. (e." 5 ." Results(of(the(Gel(filtra.on(of(the(cellulose(in( the(TLC?( Provides a solid insoluble support for the stationary phase . which is water associated with the cellulose.on( The Mobile phase is the buffer which passes through the chromatography column." " Molecules with net charges opposite to those on the ion exchange column will form ionic bonds and bind tightly to the stationary phase." " Different solvents may change the ionisation of amino acids and therefore their distribution between polar stationary and less polar mobile phases. (e. lysine and glutamic acid)" " Less polar molecules are more soluble in the organic mobile phase and migrate further. glutamate is less polar in an acidic solvent than in a basic solvent." Ion(exchange(chromatography( The Mobile phase is the buffer which passes through the chromatography column.! ! The Stationary phase is water (buffer) immobilised in pores in the gel filtration particles.on(Expt.! ! The Stationary phase is water associated with cellulose fibres.on(of(amino(acids( The Mobile phase is the solvent (n-butanol/acetic acid/ water). affecting partitioning between the stationary and mobile phases." " Larger molecules will pass through the column faster than smaller molecules (the opposite of conventional filtration).g.TLC(separa.! ! The Stationary phase is ionised groups immobilised on the ion exchange particles. " " This means that at pH values <pI." What(other(tests(might(assist(the( diagnosis?( 16 14 12 Patient 1 10 Patient 2 8 Patient 3 6 Patient 4 4 Patient 5 normal! No fast! 2 0 0 30 60 90 120 Time (min) 150 180 Urinary [glucose]" " Plasma [insulin]" " Glycosylated hemoglobin" Fasting is required to ensure a normal initial fasted blood glucose" 6 . a very high blood [glucose] after taking the glucose load. a protein will have an overall +ve charge.5 " " "+H3N-CH2-COO.5" " "+(NH2)2C-NH-CH2-CH2-CH2-CH(NH3+)-COO-" Ion(exchange(chromatography(of(proteins( Ion exchange chromatography is an important technique for separating and purifying proteins. the protein will have an overall -ve charge and bind to an ion exchange column which has +ve charged groups.! " The pI (isoelectric point or isoelectric pH) is the pH at which a protein has an overall charge of zero.cal( 16 14 12 Patient 1 10 Patient 2 8 Patient 3 6 Patient 4 4 Patient 5 2 normal! onset of diabetes! untreated diabetic! 0 0 30 60 90 120 150 180 Time (min) Someone with untreated diabetes is expected to have a high fasted blood [glucose] (time zero). and bind to an ion exchange column which has -ve charged groups. but the arginine will elute much more slowly." " At pH values >pI.Ion(exchange(chromatography( Ion(exchange(chromatography( The Stationary phase has negatively charged groups immobilised on the ion exchange particles" (-SO3-). and a blood [glucose] that has not returned to normal values after 3 h.cal( " and "+(NH2)2C-NH-CH2-CH2-CH2-CH(NH3+)-COOH" " BOTH will bind." " Glycine has no net charge at pH 5.2" " "+(NH2)2C-NH-CH2-CH2-CH2-CH(NH3+)-COO-" " At pH 2.2" " "+H3N-CH2-COO-" BINDS! " Arginine has a net charge of ~+1 at pH 5." Glucose(Tolerance(Test(Prac.0! " Glycine has a net charge of ~+0." Glucose(Tolerance(Test(Prac.and +H3N-CH2-COOH" " Arginine has a net charge of ~+1.