1.

Define the following terms:

A. Oxygenase - is any enzyme that oxidizes a substrate by transferring the oxygen from molecular oxygen
O2 (as in air) to it.
B. Epimerase- -an isomerase that catalyzes inversion of the configuration about an asymmetriccarbon atom in a substrate ha
ving more than one center of asymmetry; thus epimers are interconverted.
C. Protease - (also called peptidase or proteinase) is any enzyme that performs proteolysis, that is, begins
protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain
forming the protein.

D. Hydroxylase - any of a group of enzymes that catalyze oxidation reactions in which one of the two atoms of
molecular oxygen is incorporated into the substrate and the other is used to oxidize NADH or NADPH
E. Oxidase - is any enzyme that catalyzes an oxidation-reduction reaction, especially one involving
molecular oxygen (O2) as the electron acceptor
2. Histidine is frequently used as a general acid or base in enzyme catalysis. Considering the pKa value of the side
chain, suggest a reason why is this so?
The side chain of histidine includes the ionizable imidazole ring. The pK a value for the ring is
approximately 7.0, so at physiological pH, both the acid and base forms are present. The acid form,
the imidazolium ion, is a resonance hybrid of two practically equivalent contributingforms. Either of the two ring
nitrogens can release a proton (H+) to produce the conjugate base form, imidazole. Release of proton from the
upper nitrogen of the imidazolium ion produces the imidazole tautomer , and release of proton from the lower
nitrogen produces another imidazole tautomer. The tautomers equilibrate by way of the protonated form B. At pH
values near 7, all three forms are present in equilibrium.
Because the acid form, imidazolium, is a hybrid of the two contributors, it is plausible to show release of
a proton from either nitrogen atom in proposing a reaction mechanism involving the imidazolium form as a
general acid. Because the base form, imidazole, exists as two equilibrating tautomers, it is plausible to write either
tautomer in proposing a reaction mechanism involving the imidazole form as a general base.
Thus, histidine is frequently used as a general acid or base in enzyme catalysis.
3. How is the cooperative behavior of allosteric enzymes reflected in a plot of reaction rate against substrate
concentration ?
The sigmoidal curve indicates the cooperative behavior of the allosteric enzyme. The curve becomes more
sigmoidal in presence of activators and less sigmoidal in presence of inhibitors.
4. What is the metabolic role of of aspartate transcarbamoylase?

5. What properties of metal make them useful co-factots?

transition metals are Useful as enzymes cofactors because they have concentrations of positive
charge, can act as lewis acids, and can bind to two or more ligands at the same time

6. You are a physician with a patient who is suffering from premature pancreatic zymogen activation, which means
that the pancreatic enzymes are being acrivated in the pancreas (rather than in the small intestine) and pancreatic
tissue is being damaged as result. What would be most effective: chymotrypsin inhibitor, a trypsin inhibitor, or an
elastase inhibitor? Why?
Trypsin activates other zymogens. Consequently, it is vital that even small amounts of trypsin be prevented from
initiating the cascade prematurely. Trypsin molecules activated in the pancreas or pancreatic ducts could severely
damage those tissues, leading to acute pancreatitis. Tissue necrosis may result from the activation of proteolytic
enzymes (as well as prolipases) by trypsin, and hemorrhaging may result from its activation of elastase. We see
here the physiological need for the tight binding of the inhibitor to trypsin. Thus, a trypsin inhibitor would be
most effective in this situation.
7. A bacterial enzyme catalyzes the hydrolysis of maltose as shown in the reaction given below:
Maltose + H2O 2 glucose
If the reaction has a Km of 0.135 mM and a V max of 65 mol/min. What is the reaction velocity when the
concentration of maltose is 1.0 mM?
1/Vo = (Km/Vmax) (1/S) +1/Vmax
= (0.135mM/65 mol/min) (1/1.0mM) + (1/65 mol/min)
= 57.27 mol/min
8.

Use the plot provided to estimate Km and Vmax values of the enzyme catalyzed reactions.

9. When[S] = 5 KM, how close is vo to Vmax? When [S] = 20 KM, how close is vo to Vmax? What do these results tell
you about the accuracy of estimating Vmax from a plot of vo versus [S]?
Substrate

KM (M)

N-Acetylvaline ethyl ether

N-Acetyltyrosine ethyl ether

8.8 X 10 -2
6.6 X 10-4

10. The KM values for the reaction of chymotrypsin with two different substrates are given:
A. Which substrate has the higher apparent affinity for the enzyme? Explain.

KM is the substrate concentration at which the reaction velocity is half-maximal.

Therefore, if an enzyme has a small value of KM, it achieves maximal catalytic efficiency at low
substrate concentrations.In this case, it is N-Acetyltyrosine ethyl ether.
B. Which substrate is likely to give a higher value for Vmax?

N-Acetyltyrosine ethyl ether

11. The enzyme deoxyribokinase catalyzes the conversion of 2-deoxyribose to ADP. A study of this enzyme
produced the following data:
Exp #
1
2
3
4
5
6
7

[2-deoxyribose]
mmol/L
1.00
1.60
2.56
5.12
6.88
15.0
20.0

rate of ADP formation

(mole/10min)
0.124
0.147
0.191
0.255
0.274
0.294
0.300

Use V versus [S] and a 1/V versus 1/[S] plots to graphically determine the K M and Vmax for this enzyme.

Vmax = 0.328 (mole/10min)

Km = 1.7364

12. The following data were obtained on isocitrate lyase from an algal species. Deduce the K m and Vmax for the
enzyme, and determine the Ki for OAA and the nature of the inhibition by 0.5 mM oxaloacetate (OAA). See
solutions for info.
[S]m
0.0318
0.0464
0.0593
0.1185

rate Glyoxalate formation

(mole/10min)
0.0420
0.0583
0.0700
0.0955
0.2222 0.1167

Rate with OAA present

0.0040
0.0055
0.0075
0.0131
0.0233

13. The kinetics of an enzyme are measured as a function of substrate in the presence and the in absence of 2mM
inhibitor (I).

[S], (M)
3
5
10
30
90

Velocity (mol/minute)
No inhibitor
Inhibitor
10.4
4.1
14.5
6.4
22.5
11.3
33.8
22.6
40.5
33.8

A. What are the values of Vmax and KM in the absence of inhibitor? In its presence? In its presence?
B. What is the type of inhibition?
14. The following kinetic data were obtained for an enzyme in the absence of an inhibitor (1) and in the presence of
two different inhibitors (2) and (3) at 5mM concentration.
[S], mM
1
2
3
8
12

(mol/mL/sec)
(1)
12
20
29
35
40

(mol/mL/sec)
(2)
4.3
8
14
21
26

(mol/mL/sec)
(3)
5.5
9
13
16
18

A. Determine the Vmax and KM of the enzyme

B. Determine the type of inhibition.
11. Why is it surprising that some RNA molecules may have enzymatic activity?
RNAs form a surprisingly versatile class of molecules. As we have seen, splicing is catalyzed largely
by RNAmolecules, with proteins playing a secondary role. Another enzyme that contains a key RNA component is
ribonuclease P (RNAse P), which catalyzes the maturation of tRNA by removing nucleotides from the 5 end of the
precursor molecule. Tthe RNA component of ribosomes is the catalyst that carries out protein synthesis.
The versatility of RNA first became clear from observations regarding the processing carried out on ribosomal RNA in a
single-cell eukaryote. In Tetrahymena (a ciliated protozoan), a 414-nucleotide intron is removed from a 6.4-kb precursor

to yield the mature 26S rRNA molecule. In an elegant series of studies of this splicing reaction, Thomas Cech and his
coworkers established that the RNA spliced itself to precisely excise the 414-nucleotide intron. These remarkable
experiments demonstrated that an RNA molecule can splice itself in the absence of protein and, indeed, can have highly
specific catalytic activity.
12. Explain, on the basis of diffusion, why there is a limit to the maximum size of the turnover number of any
enzyme.
the maximum value for the catalytic efficiency can be determined by diffusion limit because as soon as enzyme
and substrate can come together it can make the products. An enzyme with 10 9 units of catalytic efficiency is very
efficient enzyme
13. Prove that Km equals the substrate concentration at Vmax .

14. Why do structural analogs if the transition-state intermediate of an enzyme inhibit the enzyme competitively and

with low Ki values (binds tightly)?

competitively because they occupy the same site as the substrate would. as for the Ki - in transition state the
enzyme makes the most contacts to the intermediate (analog in this case)
15. Inhibitors of acetylcholinesterase, such as edrophonium, are used to treat Alzheimers disease. The substrate for
acetylcholinesterase is acertlcholine. Structures are given below:

H3C

O
O
Acetylcholine

CH3
+
N CH3
CH3

H3C
+
H3C N
H3C

OH

Edrophonium

A. What kind of inhibitor is edrophonium? Explain.

Edrophonium is a quaternary alcohol that reversibly binds to the active site of cholinesterases, preventing
access & hydrolysis of acetylcholine. Thus it acts as competitive inhibitor.
B. Can inhibition by edrophonium be overcome in vitro by increasing the substrate concentration? Explain.

The inhibition of acetylcholinesterase by edrophonium can be overcome in vitro

by saturating concentrations of substrate by having more substrate available for binding.
C. Does this inhibitor bind reversibly or irreversibly to the enzyme? Explain.
Edrophonium is a quaternary alcohol that reversibly binds to the active site of cholinesterases, preventing access
& hydrolysis of acetylcholine