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Bios.

301

Name: answers

Problem Set 3 - Bisubstrate Kinetics and Glycolysis Due Wednesday, October 31, 2012, 5 PM
(Box outside Room 309, Keck Hall or in Class at 12 Noon)

Practice Problems from LPOB-14, pp. 565-566, problems1-17. Just work and understand these problems in the text -Don't turn these in! Answers are on pp. AS15 and AS16. Problems to be completed and turned in (25 points toward total class points) 1. (4 points) Although all higher organisms use the glycolytic pathway, some bacteria utilize an alternative route for glucose breakdown called the Entner-Doudoroff pathway. Provide an enzyme name for each step in the pathway and write out reasonable mechanisms with arrows indicating group transfers (i.e. Pi, H-, etc.) for the first 4 steps in the Entner-Doudoroff pathway, which is shown below. Show the key structural features of the cofactors and indicate if the sugar is in its hemiacetal or open form and explain the enzyme name.
CHO H C OH HO C H H C OH H C OH CH2OH ATP (1) kinase (hexokinase) ADP CHO H C HO C H C H C NAD+ NADH H OH (2) HO H G-6-P H OH dehydrogenase H OH O (1). Kinase reaction: H C OH H C O H H H O P OO ADP CO2(-) C OH C H H2O (3) CO2(-) C O H C H CO2(-) C O (4) CH3 +

CH2OPi

C OH dehydratase H C OH aldolase CHO C OH (like enolase) H C OH H C OH CH2OPi CH2OPi CH2OPi H C OH H C O H O P OO H

Nucleophilic attack of C6-OH on !-Pi of ATP

(2). G-6-P dehydrogenase - requires hemi-acetal sugar ring to get hydride transfer (looks like an alcohol): H O H H O H O-Pi H O-Pi HO C C HO HO NH2 NH2 HO + HO O H HO HO + N H H OH N NAD+ OH H R H R OH glucose-6-Pi NADH lactone (hydrolyzes) (3). Dehydratase - dehydration like enolase - loss of water to make an enol that isomerizes spontaneously to an "-keto acid (-)O2C O CO2(-) base: (-)O2C O C H H C OH C "-keto acid enol to ketone H O C H "-carbanion C H C H H+ isomerization to enol H+ H (4). Reverse Aldol condensatioin- like aldolase in the glycolytic pathway. active site lysine CO2(-) (-)O2C H2O (-)O2C C O C NH C NH+ H C H + NH3 H H C C Schiff's base H H C OH H-base :base H H formation H C OH H C O O CH2OPi H C D-glyceraldehde-3-Pi (-)O2C H H2O C NH+

CO2(-) H C OH HO C H H C OH H C OH CH2OPi glucuronate-6-P

(-)O2C H

C H H

C H H

Pyruvate

Bios. 301

Name: answers

2. (5 points) Rieder and Rose (1959, J. Biol. Chem. 234, 1007) performed the following experiment to examine the stereo-specificities of the three key middle enzymes in glycolysis. Dihydroxyacetone phosphate (DHAP) was labeled with tritium using muscle aldolase by incubating DHAP by itself with the enzyme in T2O. The labeled DHAP was then incubated with muscle phosphotriose isomerase, glyceraldehyde-3-Pi dehydrogenase, arsenate, and NAD+ (no ADP or ATP was added). After spectrophotometric measurements showed that the reaction was complete, tritium labeled NADH was recovered. The isolated NADH was then oxidized with acetaldehyde and yeast alcohol dehydrogenase. Of the 182,000 cpm (counts per minute) originally present in the labeled dihydroxyacetonephosphate, 167,000 cpm were recovered in the oxidized NAD+, which contains a tritium atom at the C4 position. a. (1 point) Write out the reactions described above using structural formulas for the glycolytic metabolites and include cofactors. (Less detail here is o.k. I have put some of the answers to the next page in this diagram.)
Dihydroxyacetone-P CH2OPO3
=

D-glyceraldehyde-3-P

C O pro-R HO C H H pro-S

aldolase T2O

CH2OPO3= C O HO C H T pro-R

O C NH2

TIM

CH2OPO3= HO O C C H T

pro-S

Aldolase catalyzes stereospecific removal of only the pro-S H atom

Triose isomeras catalyzes stereospecific removal of only the pro-R H atom

N NAD+ R AsO4H= instead of Pi

GAPDH (si or pro-S or B side hydride addition)


H T O C NH2 O O O AS O (-) O (-) C CH2OPO3= 1-arseno-3-phospho-D-glycerate 3-Pi-D-glycerate (-) O O C H C OH CH2OPO3=

O
CH3CH

O C NH2

ADH (re or pro-R or A side hydride removal)


OH CH3

N R

H C OH

Spontaneous, irreversible
O + HO AS O (-) O

N R

+ H C H

b. (1 point) Why do you think arsenate was added to the labeled DHAP/enzyme mixture in order to make the reaction to go to completion? Arsenate replaces phosphate in the GAPDH reaction. The mixed arsenocarboxylate anhydride spontaneously hydrolyzes to 3-Pi-glycerate, driving the GAPDH reaction to completion.

Bios. 301
(Problem 2 continued)

Name: answers

c. (1 point) From the observed data, what conclusion can be drawn about the relative stereospecificities of aldolase and triose isomerase with respect to ! carbanion proton removal and enediol isomerization? Aldolase and triose isomerase must remove different hydrogen atoms from the C3 atm of dihydroxyacetonephosphate or otherwise the radiolabel would have been "lost" from Dglyceraldehyde phosphate.
=

O3POH2C HO C H*

DHAP
C H

His95 O
Hydrolysis of Schiff's base and dissociation of DHAP
=O
3POH2C

+ Aldolase
Proton labeled with aldolase

HO

H2O

C H

C H (-) O

H N +

abstraction of C1 proton from the "top"

N versus

pro-S

Note proton is abstracted and added from the "bottom"

H H O

O C

(-)

C
=

Triose isomerase proton abstraction from the "top", Pro-R

DHAP

C H

CH2OPO3

Glu165

d. (1 point) Draw the portion of NAD+ which is involved in oxidation-reduction and indicate the position to which hydride ion is transferred.
Hydride transfer or removal from the right: B-type on pro-S side (GAPDH, glycerolPDH) Hydride transfer or removal from the right: A-type on pro-R side (ADH, LDH)

back H O C N R back H H

pro-R facing outward NH2

H O

C NH2 N R

O H2N C

pro-S facing outward

N R

e. (1 point) From the observed data, what conclusion can be made concerning the relative stereospecificities of glyceraldehyde-3-Pi dehydrogenase and yeast alcohol dehydrogenase with respect to hydride transfer to and from NAD? As described above, the two dehydrogenases transfer H- from their substrates to the opposite faces of the pyridine ring in NAD+. ADH adds and substracts the hydride atom from the A, pro-R, or re-side and GAPDH adds and substracts from the B, pro-S, or si-side (see lecture 2, p. 8).

Bios. 301

Name: answers

3. (6 points) UDP-glucose pyrophosphorylase catalyzes the formation of UDP-glucose, which is the first step in the synthesis of glycogen. Glycogen is the main storage polysaccharide in liver and skeletal muscle
O CH2OH O H O O glucose-1-P P OO+ HO O P OO O P OO O P OUTP O O N O O NH CH2OH O H O P OUDPG O O P OO O N O NH O + -O O P OO O P OOH

pyrophosphate (PPi)

In order to determine the reaction mechanism of this enzyme, the following steady state kinetic experiments were carried out.
[glucose-1-P] " 2 5 10 30 100 1000 [UTP] = 5.0 " 3.3 6.7 10.0 15.0 18.2 19.8 Initial velocity (vi) (moles/mg-min) for [UTP] = [UTP] = 20" 2,000" 8.3 16.7 16.7 33.3 25.0 50.0 37.5 KM(obs) is 75.0 45.4 unchanging 90.9 49.5 99.8
50 100

[UTP] !M 5.0 20.0 2000

KM(obs) !M 10 10 10

Vmax(obs) 20 50 100

a. (1 points) From these data, fill in the table above for the values of KM(obs) and Vmax(obs) and indicate below whether or not the mechanism for this synthase involves a ternary complex or two sets of binary complex reactions. Explain your answer in terms of the general expression for the initial velocity of a two substrate, enzyme catalyzed reaction.
The pyrophosphorylase or UDPG synthetase catalyzes the reaction by a ternary complex mechanism because the ratio KM(obs)/VMAX(obs) is decreasing. Because, KM(obs) is unchanging the binding of UTP and glucose-1-P appears to be random.

VMAX(obs) = 20

b. (2 points) From these data compute the value of the "true" Vmax and the values of the various Michaelis constants for your mechanism, i.e. Ka, Kb, and Kab.
The general equation for VMAX(obs) is: The general expression for KM(obs)is:
K M ( obs ) = [ B] V V MAX ( obs ) = MAX . You could either solve two Kb + [ B ] Kab + Ka [ B ] but since KM(obs) is unchanging Kb + [ B ]

equations in two unknowns for the data pairs in the left-hand table or assume that VMAX(obs) at 2000 !M UTP gives the true VMAX at [glucose-1-P], [UTP] = #. Using the latter assumption:
50 = 100 20 " K b = 20M K b + 20

because the mechanism of binding appears random, KM(obs) = Ka = 10 !M. The random mechanism requires that Kab=KaKb, so Kab = 2010 = 200 !M2

If the enzyme were using a binary complex mechanism, it would be picking up a UMP moiety (~Y) and release PP i (X) and then transferring the UMP group to glucose-1-Pi (Z) to from the final product UDPG (Y~Z). The exchange reactions would be (see page 8):
X~Y UTP* + E E~UMP + Glc-1-P* Z E~Y E~Y X E~UMP + PP*i E + UDP*G Z~Y

c. (1 point) Devise an isotope exchange reaction to test whether or not the enzyme is participating in a binary complex mechanism with one of the substrates (use either 14C labeled sugar or 32P). Based on your answer to Part A, will these exchange reactions occur? (hint: Lecture 3, p. 7 and 8)

Bios. 301

Name: answers

d. (2 points) Another possible mechanism for UDP-glucose pyrophosphorylase is a ternary complex mechanism with random addition of substrates and the rapid equilibrium assumption for the binding of the two substrates: A B EA
E KB KA B EB A KB KA kcat EAB Release of products P and Q

KA and KB are equilibrium dissociation constants for the binding of substrates A and B. (i). (1 point) Derive an expression for the initial velocity of UDP-glucose pyrophosphorylase, vi, in terms of [A], [B], [E]o, kcat, KA, and KB. First fill in the expressions below and then combine them. vi = kcat[EAB]
KA = K [EA] [E][A] ! [E] = A = [A] [EA] KA K B [EAB] K K [EAB] [B] = A B [A][B] [A]

[E]o = [E]+[EB]+[EA]+[EAB]

K K [EAB] K A [EAB] K B [EAB] [E]0 = A B + + +[EAB] [A][B] [A] [B] !K K $ K K E0 = # A B + A + B +1&[EAB] " [A][B] [A] [B] %

KB =

K [EAB] [E][B] [EA][B] [E][B] = ! [EB] = and [EA] = B [B] [EB] [EAB] KB K A K B [EAB] [B] K [EAB] [A][B] [EB] = = A KB [A]
1 E0 [A][B] K +[B] ' B 1 K B +[B] + K B +[B] [A] K B +[B]

[EAB] =

E0 E0 [A][B] [A][B] ' = = !K K K A K B $ [A][B] K A K B + K A [B]+ K B [A]+[A][B] K A A B + + +1& # " [A][B] [A] [B] %

) (

k cat E0 [B] E 0 [A][B] [A] K +[B] K +[B] [EAB] = B ! v i = k cat [EAB] = B ; K A +[A] K A +[A]

VMAX [B] [A] K +[B] vi = B K A +[A]

(ii). (1 point) Does this mechanism fit with the data observed on the previous page for UDP-glucose pyrophosphorylase and is the binding of the two substrates ordered or random? Yes, this expression predicts that KM(obs) should be independent of [B] or [UTP], which is what is observed on the previous page.

Bios. 301

Name: answers

4. (6 points) Citrate synthase catalyzes the condensation of acetyl CoA with oxaloacetate to form citrate and is the first step in the citric acid cycle for the complete oxidation of fatty acids and sugars.
O C CH3 S CoA + Acetyl CoA O C Citrate HSCoA synthase Citrate OH + O C CH C CH2 CO2 2 2 CO2

O2 C

CO2 CH2 Oxaloacetate(OAA)

a.

(1 point) The following double reciprocal plots were obtained when oxaloacetate (OAA) was varied at three different acetyl CoA concentrations. What does this pattern say about the mechanism of increasing [Acetyl CoA]] citrate synthase and why?
1/vi

The converging lines imply a ternary complex mechanism because KM(obs)/VMAX(obs) is decreasing with increasing [B]. Because KM(obs) is decreasing markedly, an ordered mechanism is suggested with OAA binding first.

1/[OAA]

b. Acetonyl CoA is a strong inhibitor of citrate synthase and has the structure shown to the right. Two sets of inhibition studies were carried out to examine the mode of inhibition exerted by this compound.
Fixed OAA 1/vi increasing [I]

O C CH3 CH2 S CoA

Acetonyl CoA

i. (1 point) In the first set of experiments, oxaloacetate was fixed and [acetyl CoA] was varied at three different acetonyl CoA inhibitor concentrations. Based on the pattern to the left, what kind of inhibition is observed? Explain your answer in terms of effects on KM(obs) and VMAX(obs).
A competitive inhibition pattern is observed with KM(obs) increasing and VMAX(obs) remaining unchanged. This pattern makes sense since acetonylCoA "looks" line acetylCoA.

1/[acetyl CoA]
Fixed Acetyl CoA 1/vi increasing [I]

ii. (1 point) In the second set of experiments, acetyl CoA was fixed and [oxaloacetate] was varied at three different acetonyl CoA inhibitor concentrations. Based on the pattern to the left, what kind of inhibition is observed? Explain your answer in terms of effects on KM(obs) and VMAX(obs).
This pattern describes uncompetitive inhibition because both K M(obs) and VMAX(obs) are decreasing by the same extent with increasing [acetonylCoA] so the slope of the double reciprocal plot is unchanged.

1/[OAA]

c.

(1 point) What do these inhibition patterns say about the mechanism of citrate synthase and the order of addition of the OAA and acetyl-CoA substrates?

These data show that citrate synthase catalyzes the reaction using a ternary complex, ordered mechanism in which OAA binds first before AcetylCoA (see LPOB, pp. 622-623).

Bios. 301

Name: answers

d. (2 points) Other workers have reported that citrate synthase Salmonella typhimurium shows an ordered ternary complex mechanism, which they wrote as:
E + A KA EA + B K'B EAB kcat release of products

KA and K'B are equilibrium dissociation constants for the binding of the substrates A and B. (i). (1 point) Is this mechanism consistent with the kinetic data on the previous page? If so, which substrate is A and which is B?

Yes, this mechanism specifies an ordered, ternary complex mechanism with A or oxaloacetate binding first and then AcetylCoA or B.

(ii). (1 point) Derive an expression for the initial velocity of citrate synthase activity, vi, in terms of [A], [B], [E]o, kcat, KA, and K'B. First fill in the expressions below and then combine them. vi = kcat[EAB] [E]o = [E]+[EA]+[EAB]
K [EA] [E][A] KA = ! [E] = A = [A] [EA]
K!B =

KA

K"B [EAB] K K" [EAB] [B] = A B [A][B] [A]

K! [EAB] [EA][B] " [EA] = B [B] [EAB] # K K! K! & K K! [EAB] K!B [EAB] [E]0 = A B + +[EAB] " E0 = % A B + B +1([EAB] " [A][B] [B] $ [A][B] [B] '

[EAB] =

E0 [A][B] E 0 [A][B] E0 [A][B] ) = = !B K!B KAK [A][B] K A K!B + K!B [A]+[A][B] K A K!B + K!B +[B] [A] + +1 [A][B] [B] k cat E0 [B] E 0 [A][B] VMAX [B] [A] [A] K!B +[B] K!B +[B] K!B +[B] [EAB] = v i = k cat [EAB] " v i = or v i = K A K!B K A K!B K A K!B +[A] +[A] +[A] K!B +[B] K!B +[B] K!B +[B]

Bios. 301

Name: answers

5. (4 points) The second step in the catabolism of arginine involves transamination of the $-amino group on ornithine to !-ketoglutarate (see LPOB-18, Fig. 18-26, p. 698).
CO2 CH2 CH2 CH2 NH3
+ +

H C NH3

CO2 CH2 CH2

CO2 ornithine-!-aminotransferase (E) CH2 CH2

C O +

H C NH3

CO2 + H C NH3 + CH2 CH2 CO2


-

CO2 "-ketoglutarate (B)

ornithine (A)

glutamate-5semialdehyde (P)

glutamate (Q)

The mechanism proposed for this enzyme is:


k1 E + A KA EA P + E* + B KB E*B k2 Q + E

KA and KB are equilibrium dissociation constants for the binding of orinithine (A) and !-ketoglutarate (B) to the enzyme and E* is the form of the enzyme containing a nitrogen atom from the $ -amino group of ornithine on the vitamin B6 cofactor (pyridoxamine - see LPOB-18, pp. 677-680). a. (1 points) Design a set of isotope exchange reactions to test whether or not this mechanism is correct.
Remember:

Ornithine*-NH2 + E E~NH2 + !-ketoglutarate*

E~NH2 + Glu-semi-aldehyde* E + Glu*

b. (1 points) What kind of pattern would be observed in double reciprocal plots of 1/vi versus 1/[A] at different concentrations of B? Explain your answer in one or two sentences.
1/vi
Increasing [B]

Parallel lines would be observed because for a binary complex mechanism KM(obs) and VMAX(obs) both increase to the same extent, [B]/(Kb+[B]). Thus, KM(obs)/VMAX(obs) is unchanging.

1 / [A]

Bios. 301

Name: answers c. (2 points) Derive an equation for vi in terms of [A], [B], E0, KA, KB, k1, and k2, where E0 is the total enzyme concentration and the constants are defined in the mechanism above. (i). (1 point) Write out expressions for the following:
k1 E + A KA
vi = d[P] = k1[EA] or k 2 [E * B] dt
KA =

Remember the proposed mechanism is:

k2 P + E* + B KB E*B Q + E

EA

K [EA] K [E * B] [E][A] [E*][B] ! [E] = A ; KB = ! [E*] = B [A] [B] [EA] [E * B]

E0 = [E]+[EA]+[E*]+[E*B] (ii). (1 points) Using these equations and the following steady state assumption for the modified enzyme intermediates, derive the equation for vi and find expressions for KM(obs) and Vmax(obs) in terms of [B], k1, k2, KA, KB, and Eo.
k [EA] K [E * B] d([E*]+[E * B]) = k1[EA] ! k 2 [E * B] = 0 ! [E * B] = 1 ; then[E*] = [E*] = B = dt k2 [B] KB k1[EA] k2 K k [EA] = B 1 k 2 [B] [B]

E0 = [E]+[EA]+[E*]+[E * B] = [EA] = E0

K A [EA] K k [EA] k1[EA] ! K A K k k $ +[EA]+ B 1 + =# +1+ B 1 + 1 &[EA] # [A] [A] k 2 [B] k2 k 2 [B] k 2 & " %

E0 k 2 [A][B] E0 k 2 [A][B] k [A][B] ' 2 = = ! k $ k [A][B] k K [B]+ K k [A]+ k + k [A][B] k K [B]+ K k + k + k [B] [A] K A K B k1 2 2 A B 1 1 2 2 A B 1 1 2 + + #1+ 1 & [A] k 2 [B] # k 2 & " % E0 k 2 [A][B] E 0 k1k 2 [B] k1 + k 2 k1 + k 2 [A] E 0 k 2 [A][B] VMAX [B] k1K B k1K B [A] +[B] + [B] K B k1 + k1 + k 2 [B] K b + [B] k1 + k 2 k1 + k 2 [EA] = = ; v i = k1[EA] = or k 2K A k 2 K A [B] k 2 K A [B] K a [B] +[A] [B] + [A] k1 + k 2 k1 + k 2 K b +[B] K B k1 + k1 + k 2 [B] +[A] + [A] k1K B k1K B +[B] +[B] k1 + k 2 k1 + k 2

) )

Where:
VMAX (obs) = kk E VMAX [B] kK and VMAX = 1 2 0 ; K b = 1 B K b + [B] k1 + k 2 k1 + k 2

K M (obs) =

K a [B] k K kK and K a = 2 A ; K b = 1 B k1 + k 2 k1 + k 2 K b +[B]