E12 Yeast Metabolism Postlab

E12 | YEAST METABOLISM catabolism & anabolism - we do produce ATP, and H2O, Co2, NH3 (energy-poor end products) from catabolism - exergonic (release energy anabolism requires energy (endergonic) to form large, complex molecules (buildup) from small glucose {isomerase} fructose chew gum, has fructose. skips the isomerization step, committed steps na agad. Saccharomyces cerevisiae objectives - metabolic fates of pyruvate - krebs cyvce - aerobic metabolism -

metabolic fates of pyruvate (an alphaketo acid) 2,4-DNPH (+ for alde, ket) + result: orange/yellow ppt -: orange to red solution terminal (“outside”) lone pair is the first to attack if acidic, will not attack. because saturated with H+ DNPH detects pyruvate presence. in basic (pH 8), pyruvate decarboxylase is inhibited. pyruvate is not consumed. incubate yeast in yeast, gluc in diff buffers, diff pH condition. expect to produce pyruvate…. at what condition does the pyruvate change? if pyruvate decarboxylase inhibited, pyruvate not consumed, ppt produced. 2,4-DNPH positive in blank (w gluc) only if at high temp. PYRUVATE DECARBOXYLASE ACTIVE AT LOW PH OR NEUTRAL.

effect of pH - fermentation favored when acidic. ethanol produced. o can also TCA cycle

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neutral o normal condition, TCA cycle (pyruvate consumed) - basic (high pH) o pyruvate decarboxylase inhibited TCA CYCLE succ  fumarate E: succinate dehydrogenase; FADH2 released in the expt: want to observe inhibiting effect of maleic, activating effect of phenol determined by methylene blue as a redox indicator reduced to colorless when activated if inhibited, remains blue. we look at production of CO2. when it is produced after consumption of glucpyruvate etc, CO2 gives reducing envt for methylene blue. more CO2 production = pyruvate goes into TCA (metaebolism occurs) = methylene blue becomes colorless  phenol activates. provides mildly acidic envt for solution, favors TCA. thus, CO2 produced. thus, reducing environment, so met blue reduced, becomes colorless.  maleic acid is an inhibitor of succinic acid. similar structure-wise, are analogous and thus compete for succinate dehydrogenase. o thus, only 2 CO2 formed (TCA does not proceed further) fructose decolorizes more quickly than glucose. skips isomerization part into fructose. next, PFK to F-1,6-P. rate of decolorization differs, but the color should be the same if left for a while. EFFECT OF ENERGY SRC AND TEMPERATURE TEMPERATURE 25 vs 37 vs 60 37 deg C optimal. body temp. at 60 deg, proteins get denatured. no more CO2 production. wala talaga. (at 25 deg C, more on reproduction of yeast lang ang favored. some production of CO2 (some glycolysis), but slowwww) 37 > 25 > 60 ENERGY SOURCE

glucose starch sucrose

in terms of rate glucose > sucrose > starch sucrose has gluc, fruc. starch is more diff to break down. it needs high temp, acid hydrolysis, etc, to break glycosidic bond. glucose, on the other hand, needs no hydrolysis. sucrose faster than starch. starch is basically made up only of glucose. sucrose, once hydrolyzed, has both fruc and gluc (fruc is faster), thus, paglaki ng balloon is bigger in suc compared to starch. easier to hydrolyze in the long run, sucrose has biggest balloon. it has both gluc and fruc units. EFFECT OF INHIBITORS sodium fluoride fluoride ion sequesters Mg2+ formation constant of MgF2 is higher. aagawin yung Mg2+ from the enzyme. (many enzymes are metalloenzymes) sodium citrate - inhibits PFK (phosphofructokinase) o F-6P–[PFK] F-1,6-BP COMMITTED STEP. tuluy-tuloy na sa glycolysis at this point. o allosteric regulator.  sodium citrate attaches to PFK; PFK can no longer phosphorylate F-6P CITRATE IS A BETTER INHIBITOR? DEBATABLE. NO ACTUAL TREND. JUST BASE IT ON YOUR DATA. BIGGER SLOPE, WORSE INHIBITOR. E1~E4 >> E2,3,5,6

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