Please answer all of the following.  Physical constants can be found on the back page.  Good Luck!  I hope the exam does not prove to be an April Fool’s joke.

 

1)                  A spacecraft has returned from Europa, a moon of Jupiter, where it has landed, drilled through the ice covering and recovered biological samples from the subsurface ocean.  (A plausible scenario as of current knowledge and NASA planning; I’ll believe it when I see it.)  From a small, sluglike creature you isolate an enzyme (Glycol dehydratase) which converts ethylene glycol to ethanol – i.e. antifreeze to vodka.

HOCH2CH2OH + 2H+  + 2e- CH3CH2OH + H2O

 The commercial possibilities are obvious.  The slug’s body tissues also contain an inhibitor of the enzyme.  You measure the velocity of the enzyme-catalyzed reaction at several substrate concentrations with and without the inhibitor.  The enzyme concentration used for the experiment was 1 nM.

 

[Ethylene Glycol]

(mM)

V (mM/min)

NO inhibitor

V (mM/min)

1 mM inhibitor

0

0

0

3

0.130

0.0435

10

0.333

0.1110

30

0.600

0.200

60

0.750

0.250

100

0.833

0.278

 

a)                  On the graph paper provided, make a Lineweaver-Burke plot (3 pts) of the enzyme activity data with and without the inhibitor. 

 

 

 

 

 

 

 

 

 

Lineweaver-Burke

 

What is Km for the uninhibited enzyme?      _20 uM (5 pts)

 

What is kcat for the uninhibited enzyme?     _16.7 sec-1___ (5 pts)

 

b)         Is the viral enzyme a very efficient catalyst?  Why or why not and how can you tell?  (I am more interested in the latter part than in a “yes” or “no”!)  (5 pts)

 

 

kcat/Km is a measure of efficiency, being a pseudo-first order rate constant for the catalyzed reaction.  kcat/Km = 16.7/20*10-6 = 8.35*105, which is really not very good compared to theoretical max of 108-109.

 

 

c)         What kind of inhibitor have you found (1 pt) – and why do you say so? (4 pts)

 

Effect of inhibitor is entirely on Km; Vmax is unchanged.  Therefore this is noncompetitive inhibition

 

2)                  Match the structures below to the descriptions of structure types (3 pts each):

a)   ketose              __1__

 

b)   pentose            __3__

 

c)   alduronic acid __5__

 

d)   amino sugar     __6__

 

e)   which two are anomers?        _2___ and _4___

 

e)   which two are tautomers?      _1___ and _2 or 4____

 

 

(1)

(2)

(3)

(4)

(5)

(6)

 

 

3)                  Identify the following compounds (3 pts. Each)

 

 

___adenine_____

 

___cytosine____

 

__a-D-glucose__

 

_b-D-deoxyribose___

 

4)                  Briefly describe the following: (5 pts each)

a)      Peptidoglycan

 

 

 

 

 

b)      Restriction Endonuclease

 

 

 

 

 

 

c)      Activation Energy

 

 

 

 

 

 

d)     Epimer

 

 

 

 

 


 

5)                  An explorer in Tasmania has discovered – and, of course, shot - what may well have been the last surviving Tasmanian Marsupial Tiger Thylacinus cynocephalus (there is such a creature – last known specimen died in the London zoo in the 1930’s). 

 

The animal ws a female with young in her pouch.  As a good biochemist, you of course immediately pop them all in the blender.  From the blood, you isolate the creature’s hemoglobin:  Measuring the O2 binding properties, you generate the following table of fractional binding (q) as a function of the partial pressure of O2 (pO2)

 

pO2

q

q-1

q-1q

log(pO2)

(gol)q-1q

0.3

0.00166

0.99834

0.00166276

-0.522878745

-2.779170384

0.9

0.00498

0.99502

0.005004925

-0.045757491

-2.300602467

2.5

0.0137

0.9863

0.013890297

0.397940009

-1.857288466

7.5

0.067

0.933

0.071811361

0.875061263

-1.143806841

15

0.211

0.789

0.267427123

1.176091259

-0.572794548

30

0.5

0.5

1

1.477121255

0

60

0.78

0.22

3.545454545

1.77815125

0.549671922

100

0.87

0.13

6.692307692

2

0.8255759

200

0.93

0.07

13.28571429

2.301029996

1.123384909

 

a)      Using the graph paper provided, draw a Hill plot for the Tasmanian Tiger hemoglobin (3 pts).  From the plot, determine the following parameters:

 

p50 for the T state                    158___

           

p50 for the R state                    14___

 

nH (Hill number)                     2.1___

           

minimum nbr of subunits        3___

 

(3 pts each; be as accurate as possible.  I won't penalize for being off inches, but I will for miles!)

 

b)      The protein exhibits the Bohr effect in a similar manner to human hemoglobin.  What the heck is the Bohr effect? (5 pts)

 

 

 

 

 

 

 

 

 

c)    You have also isolated and determined the sequence of two small pieces of nucleic acid from the baby tiger:

 

5’-GCCAUCUAGAUACGUG-3’

3’-CGGUAGAUCUAUGCAC-5’

5’-AUCAUCUAAAUACGUG-3’

3’-UAGUAGAUUUAUGCAC-5’

 

                                          i.    Which of these two fragments will have the lower Tm (that is, denature or have the strands separate at the lowest temperature)? (1 pt)

 

 

                                         ii.    Why? (3 pts)

 

 

 

 

 

                                        iii.    We mentioned three forms of double helix that can be formed by nucleic acids.  Which type of double helix will these molecular form and why? (4 pts)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Useful Physical Constants

 

Constant

Value

Avagadro’s Number

6.02 * 1023

Boltzmann’s Constant

1.3807 * 10-23 JK-1

Charge on electron

-1.602 x 10-19 coulomb

Gas Constant (R)

8.314 JK-1mol-1

Faraday’s Constant

96,485 JV-1mol-1

k = 1/(4pe)

8.99 x 109 Nm2coulomb-2

ln(x)

2.303 log(x)

Dielectric constant of water

78.54

 

 

 

Because I am feeling particularly warm and fuzzy this week, I will remind you that a Hill Plot plots log(θ/1-θ) versus log pO2.