Answer Key
Biochemistry Answers Problem Set II
-
University:
Amherst College -
Course:
BIOL 303 | Molecular Neurobiology: CURE Academic year:
2000
-
Views:
101
Pages:
13
Author:
Makhi Levine
Spring 2000
Biochemistry 30
Answers Problem Set U
L Enzyme Catalysis (30 points)
Shown below are three different substrates for the enzyme chymotrypsin
.
,
*
.LXU
-
-
a) Write down and label product l and product 2 for the amide bond hydrolysis for each
substrate. (6 points)
{
rodvcAl
VmdixA- 2 <6
0
MtK
s&sWM
sdoskakC
/
tffjj- CJfc C-
Q
Jl
^
SiWVI
(Substrate B),
b) The Km‘s for the three different substrates are 31 mM (Substrate A), 15 mM
the E and 5
and 25 tnM (substrate C). If we assume that the ES complex is in equilibrium with
the
starting material, we can calculate the binding energy directly from the Km. What are
rise lo
binding energies for each of the ES complexes at 37C? What interactions might give
these differences? (S points)
J
frrnfrt j
Km'
/
fQ
3tmH
&
C
iSrfH
Z 5W|
A&~'
-
~
Z
TheHjh
^
-
y
{&**.**
t
5
V 3
c) The Ic^ for substrates a, b and c are: 0.06
0.14 s'1, and 18 s'1 respectively. What is the
cdviation barrier for cadi reaction at 37“C Why do you suppose these barriers are BO
different? (8 points)
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H vnfflMt f - idtotr? m MmUdtcU d&vn stJtf
id & fifs
c*
lOrfL
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t
f l g j&toArfa P / mt - jpc /
/ i Sam? -ford ) The
/Km values help to evaluate which substrate workrTfesT with an enzyme, Which of
the substrates is the “best” substrate for chymonypsin and why , (8 points)
af
iAvailed *n
^
A
fecaJt/ Km
\M
isoocst
sdhdorcAt
^
6 1.33
ptef / y
c* 111
hesh substrate. - tneerpwaf !
iSfc vnW 4e m f t c
yod
sUtetrcftL*
/
^
ytJIt
ffed binding ( not *‘
Ittp
O
1
3
fmc»M HmutfiM
+ JJP
( njcKttt
-
lJ «« phasp*UH + ADP .+ H*
4
3
6
7
£G
+M
-
ec*
+ ATP
1
iff "
ihr4R>Ky*«tpne
photphne
-
=
*
*»»>«phaiQCuEi
,
=
Glyc»r»Wehyd* J pfioiiihii# + P + NAD ^
IJ-ttipSwpftogtyrtrau VNAOM + H *
;
U «pimjpb(»gkitfiirTO Hh ADP
JrPfmplwpImraM
=
-«
J-SJha»p*w6lYBPM
8
Thau
3-pNwphii*
&
+S 7
0L3
+ iJt
* Q ,S
+1J
--
.
c
iu(n* rtit
Giycer* Idthydt 3* pHoij!li*l«
CBhYdragtnne
PhatpIvog yGBnte Untie
^
ATP
-1A
CL
J>
«lP
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+1.7
+04
*** + ATP
OL
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S3
04
+ 0J
Clj
+
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known
I , Next to each of the reactions above, write one of the following letters which best describes
the type of chemical reaction catalyred : ( 10 points)
a Phosphoryl transfer
b Phosphoryl shift
c fsomerization
d Dehydration
e Aldol Cleavage
f Phosphorylation coupled to oxidation
2 . In each of the reactions cited above, the free energy for the reactions written under actual
cellular conditions is much more negative than the free energy under biological standard state
conditions (pH 7, 37 C ). Why is that? (4 points)
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3 Give m example &f an enzyme we have studied which exhibits the following phenomena and
o
draw 4 difeitlicil Structure fl-f if* nrtntial hinl
(16 points)
r
U
a) binds a six caibon sugar kelo in its open form
g)
flM -
b) binds to many different hexosc sugars
l- li
5c H
-
Fi't
m^,
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Dhm> bn*iSc
(5) pke'.ptvfrvc- / „ ^
(
a
-Ofl
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u
c ) is inhibited by high levels of 2,3 Diphosphoglyceraic
,
fc ; 1
I
r
/
ft*
d ) needs to bind a second molecule of oxidized substrate before it can release product
(J g ( yceraldehyde. Zphosph< ] VYM*
*200
i
<1
0
5»
0
*«.. 3H
+ 211
*167
as
33
79
3a
3.5
7
.
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*
Ct
-
15
59
»
ev
H' jljcetitt
---«
19»
VI
77
153
2560
+ 2«
* 151
*«s
71
Shown above are data for concentrations of glycolytic intermediates in a resting animal
(intial ) and in one derived of oxygen for 25 seconds (ischemia )
7
^
yPSP
^
I ) Given the concentrations of the glycolytic intermediates, and the Kins noted ai^we identify
the degree of saturation of each of the enzymes of glycolysis.
a ) in the resting animal
(
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b) in (he ischemic animal
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^^Kffiow do Allosteric effectors play a role in regulating the flow through glycolysis in an animal
suffering from ischemia?
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- Exams
2000
I . Enzymes of Glycolysis and CiEnc Add Cycle
prior to Spring
la} The reaction* catalyzed by the ten enzymes of glycolysis can be chemically classified into
the five following groups. Whai is the general name for Bn enzyme which catalyzes this kind of
chemical reaction and which enzymes of glycolysis fall into these categories. Answers for the
first group have been provided as a guide.
CLASS
ENZYMES
oxidation .
dehydrogenases
Gly 6 or GAT* dehydrogenase
phosphOTyllation :
Gty-1 or hexokinase
kinases
Gly 3 or phosphofruclokinasc
Gly- 7 or 3PG kinase
Gly 10 or pyruvate kinase
;
,
Gly- 2 or phosphoglucoisomenkse
rsomernse mutases
isomerization
Gly - 5 or tnosephosphate isomearase
Gly 8 or phosphoglyccrate mutase
enclose
Gly-9 or 2- phosphoglyceratc cnola &c
dehydration
Gly-4 or aldolase
aldolase
aided condensation:
-
-
-
,
lb) In as concise and compact prose ns possible, compare the different mechanism* used by the
enzymes which catalyze isomenzations. Identify catalytic-ally active side groups and predict
how the activity of the enzyme might vary with pf [.
-
Gly 5* TIM* catalyzes ihe conversion of DHAP to GAP, a ketone to an aldehyde, via a lLcts enediotL' intermediate. Glu 165 acts as a base to abstract a dissociable proton from the carbon
adjacent to the carbonyl, and then acts as an acid to deliver it back to the carbonyl carbon,
Ibrcing pratonaunn of the oxyanion via His 95 . The pH profile for the conversion of DHAP to
GAP L$ shown below, high rate require* a de protonated g!u and a protonated His. [Gly-2 similar
mechanism }
-
Gly 8, pliosphoglyerate mtilase, catalyzes the conversion of 3PG lo 2PG, via a H1S H1S duo at
the active site. Fhosphory1 lation of the active site His is a prerequisite for the reaction* and the
isomerization proceeds by His A abstracting a phosphate group from 3PG, at the same lime that
the osygen at postion 2 oF 3PG is abstracting the phosphate from HisB Product dissociation is
followed by an internal rearrangement where phosphate group is handed from HisA !o HisB A
lysine at the active site pocket is necessary !o Lon pair with die caiboxlyate of the substrate.
High catalytic rates are mainained by a deprntonaied Hts and a protonated Lys. can fa
^77
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s
2. SduiT base formation is « common catalytic strategy used by proteins wtuM interacting with a
carbonyf ecotaming substrate. Which of the following statements knot true regarding Schiff
base formation in aldolue. Explain
-
a) The Stuff base serves to anchor the substrate is the binding pocket by forming A
covalent enzyme-substrate adduct.
bj The Schiff base activates FBP for bond cleavage by petndding electrons to stabilize
the clcctrophillic intermediate.
c) The nitrogen used in $chi£f Base formation is derived from aLys side group,
d) Proteins employing Schiff base mechanisms should show greater activity at higher
(
PH.
3, Identify by name an enzyme wc have studied which:
(sometimes mere than one correct answer exists, just list one)
y , =. nof yet Stated
in the decarboxylation of its substrate
4£~a) uses a cofactor to bind to the substrate and assist ,
mi .
by acting as an electron sink
pyrovate. dehydwehPSZ i
h ) uses a lysine side group to form a Schiff Base intermediate which binds and activates
-
its substrate
c) uses a metal ion to activate a water molecule and form an acyl enzyme intermediate
}
Cdr &cyypipfrdciSiZ *
enzyme
d) uses a histidine side group in catalysis and forms a phosphoiylated
,
pkosphagtyazvGU wHtise ,
intermediate
,
sweet* ate thwki ^ttse,
e ) uses a cysteine side group as a nucleophile and forms a thiocster intermediate
d & bycfr&je r* A &&
product
(fc. d ) turns an achiral substrate into a chiral
{
.
e) scrambles the C° label on acctyl CeA: HjC -C-CoA
Awni /flie
-
+
foma
e which binds ATP but is
j(r f ) is a trausmembrane protein in the mitochondrial membran
not a kinase
—
frTP SyriMetosz
g) under normal cellular conditions operates below its V
glt / eolfrvisG
BAP
j
tiehytlrGgGfX&t.
u
h ) under normal cellular conditions operates at or near its V**,
he & k *** & *
i) exists m*at least two different isozymes
X-Otf
the
j) is not really an enzyme but a protein which acts as a mobile carrier of electrons in
election transport chain
Cyho rowc <
via a four electron transfer
4t k) needs to reduce its substrate
C y f . C Deft 'S*
11
l ) incorporates a radiolabeled oxygen from O water into its substrate in glycolysis
v
t-kart forws Schttf btiSfi
$ ny4tiir
in the TCA cycle
substrate
O
its
into
water
oxygen
from
"
m ) incorporates a radiolabeled
rt
aco*‘ 4a ¥. , { i / \atQsc
^
-
^
^
.
m ^
rf
,
4. fa trying to eluddato the mechanism rrfTH ea xsrimcMte were exmducted with ,
vn n j a
yocrtkktydc 3 pbosphate (GAP]flatted aid TThc dQ dm BcdoDapitepM (DHAf) n testfT
product had lost the Label at the middle carbon and showed Less thin 5% libelling tt the Cl >
~
, :.V
carbon. Which of the following is trite. Explain, JJV
Ki
*, K
'J
^^
~
^
?
^ ^
->£,
^ ^
^
-
W. •
' i *'
experiment
)
suggests
a This
that a single catalytic group on the enzyme could art be
responsible fordepertonatioa atC2 *nd subsequent repnotonation at Cl ,
'
it
,
"
JV ;::
,
( V) TMs experiment suggests thata single catalytic group on the protein is responsible for
depo& tonation and subsequent tepfutonation and has the opportunity to exchange trithum
with the solvent
c) This experiment suggests tiiat two basic groups in close proximity must shuttle the
tritunm from one carbon to another ,
5. In converting glucose to pyruvate in glycolysis* the substrate is oxidized from an a] do sugar to
a a koto acid. Which cf the statements below best describes the oxidative processes in
glycolysis. Explain,
a) oxidation occurs in gly l , gly 3* gly 6,
b) oxidation occurs in gty*I and gly-3 .
occurs in gly 6.
fcllj) oxidation
none of the above.
-
-
-
-
6, In testing the catalytic mechanism of gly-8, phosphoglycerate mutesc, mutant enzymes were
prepared with each of the possible mutations below:
a) Ht5A ut the bindingfactive site was changed to Arg,
b) Hisa at the bindmg/activc site was changed to Tyr.
Lys at the bindmg/active site was changed to Gly.
Which sample is most likely to have the same
isomerization of 3-pbosphogtyceratc? Explain.
and very different K* fur
7. Km/S values are used to evaluate the "degree of saturation" of an enzyme with a particular
substrate under particular cellular conditions. Underline the correct word i n parentheses in each
of the sentences below and choose one enzyme we have studied for which the statement is true.
a) Km/S values (greater orJcSs) than one mean that the enzyme is working at full speed
and that the rate of the reaction will not change rapidly with changes in the substrate
concentration.
-
enzyme: A&iokwasc.
b) Km/S values (sJTEffTe of less) than one mean that the engine is working far below its
Vraax and that Lhe rate of the reaction will change drastically with small changes in the
Jenzyme concentration. ~
enzyme:
as
^
GAP deAyrfrcgg^
-
r
*
>
1
* Ansu&S '
-
-
.
^ ^ ^
^
- ini iv
- to ' *ri
pwtfv afe < .
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8. Enzyme CetalytiljF*
gj >"Y
^
}
treating
ptcteaseJua
HTV 'M rceav!
The enzymeIBV4
boeo tire the most vuooeaffbllaQnt for
M
'
LofccHiotL When oombtaed with i reverie transerijXiisa inhibitor, this potent «TcttifP* his +v j hc^ ; i
^ ^
brought patients beck fetra. deaths door and restored them to full “health”, though the capacity
of the virus to "hide ouT in the CN5 for many years has meant that no one can yet c aim that
L
^
1
they are cured
,’
^
*
' «4'
The protease is an endoprulcasc that cleaves the peptide bond on the amino terminal side of the
amino add proline. la a scries of experiments on various peptide substrates, the following
catalytic constants were measured. In the table below, a dash represents the scissEle bond
cleaved by die protease.
,
x'nr,
--PVQ
Substrate ,
W*
( 4> GNY
( EO RNF-PVA
( C ) LAA-PQF
c$ LNUPVA
Km (mM)
tealfs4)
bcai/Km (mhfV1)
0.60
1.25
0.13
2.4
0.8
0.02
2.2
4.0
0.6
14.6
110,0
1.9
Assume a simple mechanism, where k
^
E + So^ ES
= kh and
= ft
_
E+P
1 , Sketch a reaction coordinate diagram indicating the heights of the activation barriers; for the
£££ J
- gT-Jvj ( /f krrty
four substrates from the data above.
*
<5 T
--
f
7Q
-
2 . What mforraation about the enzyme binding site can you get from the various Km values?
" P" 6Ws wesf MV* ; (3 tesU tffitfu
tfrs St*»«K 4L
a^_ £-$ r‘Vf*4nc &#\ fo' 3.
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3 How would you interpret the kcat/Km values in the last column 7
r
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£n*Qi*xz tJctfd effect D"
4. One inhibitor, L69S 502. which is in phase 01 clinical dials is shown below . This drug is
thought to be a competitive inhibitor . How, if at all, would the presence of B competitive
inhibitor change the experimentally derived constants in tire table above ?
-
/
A
L4 StfiQ%
* 0 1 nM
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Problem III coat'd
JM
CSph e**)
v
-
*
v. .
1. Prom the information on the previous page , what Is the k
for hydrolysis of acetylcholine by acetylcholine esterase at
-
25flC
-£ air * £St eoa sr i
How long does each acetylcholine substrate molecule stay
attached to the enzyme?
2m
{£&*!' * *fo£t$ec
*
*
3*
If the mechanism is a simple one such as discussed in
class ( equilibrium of E and s, or steady state of E and fi)
what would be the activation barrier for the rate determining
-
AG* . il (r ~ ( - 3& I,
~
HO
4, Once the initial rate of the hydrolysis has been measured
at several different concentrations of substrate, how is
calculated?
S.
^
ftjtacW
-
What would the Lineweaver Burke plots look like for the
case of:
aj the inhibition of hydrolysis of acetylcholine
protonated tertiary amine? Label all axes and lines.
by a
b) the inhibition of hydrolysis of substrate (2] by a
protonated tertiary amine? Label all axes and lines a
6,
_
JULU QjbLQXktd
and
What happens to V
SCL
cdbLacLtd
Ka
in each of these circumstances.
*7 M Does the inhibitor bind at the active site or doesn't it?
Can you offer a possible explanation of the contradictory
data?
$JLL $ &.du£
The end
M
.
l
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