Chem 128 Introduction to Chemical Biology

Van Vranken

Problem Set 7 - Key

Exercise:
Cytochrome P450 enzymes oxidize foreign molecules in the human body. Open the crystal structure for
Cytochrome P450 bound to the substrate camphor (PDB code: 3WRI). When you look at the
introductory page you can see that the ligand camphor is abbreviated as CAM in the structure and the
heme ligand is abbreviated as HEM.
A. Show the heme ligand and the camphor ligand
B. Identify the protein residue and number bound to the iron atom.
C. Display the distance from iron atom to the closest carbon atom in the camphor substrate.
D. Draw a chemical structure of camphor and identify the carbon atom that would be oxidized by the
heme to an alcohol (i.e, is closest to the iron atom in the heme).
INSTRUCTIONS
Open the structure for 3WRI in the Protein Databank
View Using JSmol
Right click with the mouse on the protein display window and click console
Select all
Cartoon off
Spacefill off
select within(4, iron)
wireframe 0.3
color cpk
(hover over the atoms to see the residue name, number, etc)
select iron
spacefill 0.7
(double left click on the iron and then double left click on any other atom to display the distance)

H
[O]

Chem 128 Introduction to Chemical Biology

Van Vranken

1. The drug captopril is an inhibitor of angiotensin I-converting enzyme. Access the crystal structure of
human testicular angiotensin I-converting enzyme bound to captopril (PDB code: 1UZF). When you
look at the introductory page you can see that the ligand is abbreviated as MCO in the structure.
-O

-O

SH

captopril

N
captopril
+
enzyme

S
X

Zn X
X
?

A. Show the captopril ligand and the Zn atom

B. Identify the protein residues bound to each zinc atom and label them with one-letter code and amino
acid residue numbers.
C. Render a picture of the ligand, Zn atom, and the residues that are covalently bound to the zinc atom.
D. Draw a chemical structure of each zinc atom and the four side chains bound to zinc.
View Using JSmol

Right click with the mouse on the protein display window and click console
Select all
Cartoon off
Spacefill off
Wireframe off
Select zinc or MCO
Wireframe 0.3
Select within(7, zinc)
Wireframe
Select within(7, zinc) and *.ca
Label %m%r
Select all
wireframe off
Select MCO or 383 or 387 or 411 or zinc
Wireframe 0.3
Color cpk
Select zinc
Spacefill
Select protein
Select mco
Dots
Select all
Rockets

Chem 128 Introduction to Chemical Biology

-O

H387

S
Zn N
N
O

H383

Van Vranken

N
H

NH

O
E411

2. Several commercial biocides were tested for lethality against aquatic plants in both fresh
water (FW) and salt water (SW).1
a. Estimate the 50% lethal dose (LD50) for Diuron against saltwater plants.
b. Which biocide the most safe to freshwater (FW) aquatic plants?

all of them
are >99% lethal
above 10,000 g/L
Diuron-SW

40 g/L
(an estimate is OK)

Atrazine (circles) is the least toxic at 1 g/L

3. Using structures in the protein databank, correctly match the protein domains with the
proteins that contain them.
Protein Databank code
3EMH
2NZI
1XIO
3FYL
3GU4

Protein Domain
Seven-transmembrane domain
kinase
Zinc finger
WD40
Fibronectin FNIII

Chem 128 Introduction to Chemical Biology

Van Vranken

Answer
Protein Databank code
3EMH
2NZI
1XIO
3FYL
3GU4

Protein Domain
WD40
Fibronectin FNIII and Ig domains
Seven-transmembrane domain
Zinc finger
kinase

4. The enzyme hexose-6-phosphate mutarotase (PDB code: 2CIR) binds glucose-6-phosphate

(ligand name BG6) more tightly than glucose. Which residues in the active site make contacts
with the phosphate group?
Answer:
View Using JSmol
Right click with the mouse on the protein display window and click console
Cartoon off
Select BG6
wireframe 0.2
select within(7,phosphorus)
wireframe 0.2
color CPK
(hover cursor over the atom to see the residue name and atom number)
(double left click an atom and when you double left click a second atom, the distance will
appear)

Gln81

Arg86

H
H
H
Arg57

H
N

N
N

N H
N H
H

O
H
O
O P O
O

H N

O
HO

H
OH
OOH
H

5. A monoclonal antibody against nitrotyrosine residues has been created for use in ELISA
assays.2 In order to test the specificity of the antibody, a variety of tyrosine deriveatives were
tested for their ability to competitively INHIBIT binding of the anti-nitrotyrosine antibody to the
nitrotyrosines on the surface of the ELISA well.
4

Chem 128 Introduction to Chemical Biology

Van Vranken

competitive inhibitors

X
HO

X
OH
HO

X
HO

OH

antibody

X
X
HO
X
HO

R
R
HO

NO 2

O2N
HO

NO 2

OH
HO

NO 2

HO

NO 2

HO

NO 2

bottom of ELISA well

a. Draw the chemical structures of 3-nitrotyrosine, 3-chlorotyrosine, and the tetrapeptide

GlyGlyTyr(NO2)Arg.
b. Based on the dose-response curves, which small molecule binds most tightly to the antinitrotyrosine antibody.

c. The protein bovine serum albumin (BSA), an inexpensive protein from cow blood, was
nitrated such that each protein had three to six nitrotyrosine residues per protein. What
percentage of the tyrosines in BSA were modified in the nitration reaction.
SQ

SEQUENCE
MKWVTFISLL
DEHVKLVNEL
ERNECFLSHK
ANKYNGVFQE

607 AA; 69293 MW; 45F4D04A CRC32;

LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA
TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD
DDSPDLPKLK PDPNTLCDEF KADEKKFWGK YLYEIARRHP
CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG

FSQYLQQCPF
MADCCEKQEP
YFYAPELLYY
ERALKAWSVA

Chem 128 Introduction to Chemical Biology

RLSQKFPKAE
CCDKPLLEKS
HPEYAVSVLL
LGEYGFQNAL
NRLCVLHEKT
DTEKQIKKQT
STQTALA

FVEVTKLVTD
HCIAEVEKDA
RLAKEYEATL
IVRYTRKVPQ
PVSEKVTKCC
ALVELLKHKP

Van Vranken

LTKVHKECCH
IPENLPPLTA
EECCAKDDPH
VSTPTLVEVS
TESLVNRRPC
KATEEQLKTV

GDLLECADDR
DFAEDKDVCK
ACYSTVFDKL
RSLGKVGTRC
FSALTPDETY
MENFVAFVDK

ADLAKYICDN
NYQEAKDAFL
KHLVDEPQNL
CTKPESERMP
VPKAFDEKLF
CCAADDKEAC

QDTISSKLKE
GSFLYEYSRR
IKQNCDQFEK
CTEDYLSLIL
TFHADICTLP
FAVEGPKLVV

Answers:
a.
HO

NO 2

HO

Cl

HO

ArOH or ArOThe pK a will be

close to 7.
+H

O-

3N

3-nitrotyrosine

NO 2

ArOH or ArOThe pK a will be

close to 7.

+H

O-

3N

H
N

H 2N
O

3-chlorotyrosine

O
N
H

H
N

O-

GlyGlyTyr(NO 2)Arg

NH
+H

2N

-6

NH 2

b. The tetrapeptide GlyGlyTyr(NO2)Arg binds most tightly, with an IC50 around 10 M.

c. I count 20 tyrosine residues, so only 15-30% of the tyrosines were modified in the nitration
reaction.

Th. Caquet, M. Roucaute, N. Mazzella, F. Delmas, C. Madigou, E. Farcy, Th. Burgeot, J.-P.
Allenou, R. Gabellec Risk assessment of herbicides and booster biocides along estuarine
continuums in the Bay of Vilaine area (Brittany, France) Environmental Science and Pollution
Research 2013, 20, 651-66.
2
Jamshad KHAN, David M. BRENNAN, Nicholas BRADLEY, Beirong GAO, Richard
BRUCKDORFER, Michael JACOBS 3-Nitrotyrosine in the proteins of human plasma
determined by an ELISA method Biochem. J. 1998, 330,795801.