CHEN382_SP17 Problem set 4 KAO

1. Decarboxylation0of0glyoxylate0(S)0that0occurs0in0the0mitochondria0is0inhibited0by0
malonate0(I).00Using0the0following0initial0rate0data0obtained0in0batch0experiments,0

determine0the0following:0

Glyox,0S0(mM)0 Rate0of0CO20evolution,0v0(mmoles/LJhour)0

0 [I]0=000 [I]0=01.260mM0 [I]0=01.950mM0

0.250 1.020 0.730 0.560

0.330 1.390 0.870 0.750

0.400 1.670 1.090 0.850

0.500 1.890 1.300 1.000

0.600 2.080 1.410 1.280

0.750 2.440 1.820 1.390

1.000 2.500 2.170 1.820

a. What0type0of0inhibition0is0this?0

b. Determine0the0constants0Vm,0Km’,0and0KI.0
0

Answer:0

0 0 0 0

4.000 0.980 1.370 1.790

3.030 0.720 1.150 1.330

2.500 0.600 0.920 1.180

2.000 0.530 0.770 1.000

1.670 0.480 0.710 0.780

1.330 0.410 0.550 0.720

1.000 0.400 0.460 0.550

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CHEN382_SP17 Problem set 4 KAO

0
2. Amyloglucosidase0from0Endomycopsis+bispora0is0immobilized0in0a0very0small0

polyacrylamide0gel0beads.00The0activities0of0immobilized0and0soluble0enzyme0are0

compared0at080ºC.00Rate0data0are0measured0at0a0fixed0substrate0concentration0with0

the0following0results.00Determine0the0halfJlife0of0each0form0of0enzyme.00Assume0

υ ~ Vm .000

0 Enzyme0Activity0(µmol0mlJ10minJ1)0

Time0(min)0 Soluble0enzyme0 Immobilized0enzyme0

00 0.860 0.450
30 0.790 0.440

60 0.700 0.430

90 0.650 0.430

150 0.580 0.410

200 0.460 0.400

250 0.410 0.390

300 J0 0.380

400 J0 0.370

3. αJAmylase0from0malt0is0used0to0hydrolyze0starch.00The0dependence0of0the0initial0

reaction0rate0on0temperature0is0determined0experimentally.00Results0measured0at0
fixed0starch0and0enzyme0concentrations0are0listed0in0the0following0table.00R0=01.990cal0

gmolJ10KJ1.00Assume0 υ ~ Vm .0

0
Temperature)(ºC)) Rate)of)glucose)production)(mmol)m83)s81))

) )

200 0.310

300 0.660
400 1.200

600 6.330

a. Determine0the0activation0energy0for0this0reaction.0

b. αJAmylase0is0used0to0break0down0starch0in0baby0food.00It0is0proposed0to0

conduct0the0reaction0at0a0relatively0high0temperature0so0that0the0viscosity0is0

reduced.00What0is0the0reaction0rate0at055ºC0compared0with025ºC?0

c. Thermal0deactivation0of0this0enzyme0is0described0by0the0equation:0

kd = 2.25 ×10 27 e−41,630/RT

0

where0kd0is0the0deactivation0rate0constant0in0hJ1,0R0is0the0ideal0gas0constant0in0

cal0gmolJ10KJ1,0and0T0is0temperature0in0K.00What0is0the0halfJlife0of0the0enzyme0

at055ºC0compared0with025ºC?00Which0of0these0two0operating0temperatures0is0

more0practical0for0processing0baby0food?00Explain.0

0
0

CHEN382_SP17 Problem set 4 KAO

4. Enzyme0E0catalyzes0the0reaction0S0!0P0and0has0a0Km’0of050x010J50M0and0a0Vmax0of0250
µM/min.00Compound0I0is0an0inhibitor0for0enzyme0E.00There0is0only0one0binding0pocket0

on0 the0 enzyme0 that0 can0 bind0 both0 the0 substrate0 and0 inhibitor.0 0 A0 reaction0 was0

started0with010x010J70M0of0E0and010x010J40M0of0S.00What0is0the0percent0reduction0in0the0

initial0velocity0of0the0reaction0(S0!0P)0if040x010J40M0of0I0were0added0to0the0reaction?00

The0KI0is030x010J40M.000

5. An0inhibitor0(I)0is0added0to0the0enzymatic0reaction0at0a0concentration0of01.00g/L.00The0

following0data0were0obtained.00The0Km’0=09.20g0S/L0in0the0absence0of0inhibitor.0

0
v0(g0LJ10hJ1)0 [S]0(g/L)0

0.9090 200

0.6580 100

0.4930 6.670

0.400 50
0.3330 40

0.2890 3.330

0.2270 2.50

a. Is0the0inhibitor0competitive0or0noncompetitive?0

b. Find0KI.0

6. Protease0 (an0 enzyme0 that0 breaks0 down0 proteins)0 is0 being0 inactivated0 by0 UV0 light.00

The0 deactivation0 is0 assumed0 to0 be0 a0 first0 order0 reaction0 with0 deactivation0 rate0
constant0 =0 0.050 minJ1.0 0 A0 protein0 degradation0 reaction0 was0 started0 with0 10mM0 of0

enzyme0 and0 1000 mM0 of0 substrate.0 0 The0 catalytic0 constant0 for0 the0 reaction0 is0 0.030

minJ10 and0 the0 Michaelis0 constant0 is0 50mM.0 0 0 After0 10 hour0 of0 UV0 irradiation,0 how0

much0undegraded0protein0(substrate)0is0left?000

7. An0enzyme0ATPase0has0a0molecular0weight0of050x01040daltons,0a0KM0value0of010J40M,0

and0 a0 k20 value0 of0 1040 molecules0 of0 ATP/(min*molecule0 of0 enzyme)0 at0 37ºC.0 0 The0

reaction0catalyzed0is0the0following:0

ATP0(with0ATPase)0!0ADP0+0Pi0

0 which0can0also0be0represented0as:0

E0+0S0⇔0ES0!0E0+0P0

where0 S0 is0 ATP.0 0 The0 enzyme0 at0 this0 temperature0 is0 unstable.0 0 The0 enzyme0
inactivation0kinetcs0are0first0order:0

[Ea]0=0[Eo]exp(Jkdt)0

where0[Eo]0is0the0initial0enzyme0concentration0and0kd0=00.10minJ1.00In0an0experiment0

with0 a0 partially0 pure0 enzyme0 preparation,0 100 µg0 of0 total0 crude0 protein0 (containing0

enzyme)0is0added0to0a010ml0reaction0mixture0containing00.020M0ATP0and0incubated0at0

37ºC.00After0120hours0the0reaction0ends0(i.e.,0t0!0∞)0and0the0inorganic0phosphate0(Pi)0

concentration0is0found0to0be00.0020M,0which0was0initially0zero.00What0fraction0of0the0

crude0protein0preparation0was0the0enzyme?00Hint:00Since0[S]>>Km,0the0reaction0rate0

can0be0represented0by:0

0 0 d[P]/dt0=0k2[Ea]0
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CHEN382_SP17 Problem set 4 KAO

8. Show0why0d[P]/dt0=0k2[Ea]0when0[S]>>Km.00
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9. Lipase0is0being0investigated0as0an0additive0to0laundry0detergent0for0removal0of0stains0
from0fabric.00The0general0reaction0is:0
0
Fats0!0fatty0acids0+0glycerol0
0
The0 MichaelisJMenten0 constant0 for0 pancreatic0 lipase0 is0 50 mM.0 0 At0 60ºC,0 lipase0 is0
subject0 to0 deactivation0 with0 a0 halfJlife0 of0 80 min.0 0 Fat0 hydrolysis0 is0 carried0 out0 in0 a0
wellJmixed0batch0reactor0that0simulates0a0topJloading0washing0machine.00The0initial0
fat0 concentration0 is0 450 gmol0 mJ3.0 0 At0 the0 beginning0 of0 the0 reaction,0 the0 rate0 of0
hydrolysis0is00.070mmol0LJ10sJ1.00How0long0does0it0take0for0the0enzyme0to0hydrolyze0
80%0of0the0fat0present?0
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