Kim D.

Terrizzi
CHEM2331 Bioanalytical Chemistry
Experiment 2: Modeling
Lab Partners: N/A
October 6th 2016
INTRODUCTION
The same enzymes appear in organisms that require overlapping metabolic needs, and
these needs are often very similar in all organisms. Lactic dehydrogenase (LDH) is an enzyme
that reduces pyruvate to lactic acid with the assistance of NADH cofactor. It appears in nearly all
cells. In the case of this lab, LDH crystallography models from the dogfish shark and Bacillus
stearothermophilus, or an extremophile species of bacteria, are studied in the DeepView
program. Based off of its distance and orientation to a corresponding substrate, any active-site
amino acids can be determined on DeepView. Additionally, because DeepView has the ability to
display multiple proteins simultaneously, the structural differences in two enzymes before
preparing for substrate binding and after can be compared, as well as those subjected to
evolutionary nuances. Homology modeling is essential for studying the inner workings of
proteins on a molecular level, providing structural and functional insights that contribute to an
overall understanding of catalysis, adaptation, performance, and reactions. This makes programs
such as DeepView a valuable tool in biotechnology fields.
While the Michaelis-Menton constant responds to temperature fluctuation by increasing
with higher temperature and decreasing with lower temperature, it can also remain constant if an
enzyme adapts. In doing so, the catalytic efficiency of organisms that live in low temperatures
must increase. This is made possible by amino acid substitutions, wherein residues are
substituted to promote greater flexibility in cold temperatures. These residues replace amino
acids that would become too rigid while submitted to lower temperatures.
Residues are often substituted in the immediate surroundings of active sites, whereas the
amino acids in the globular center are often more tightly bound, thus remaining unchanged. This
is due to the need for flexibility on the surface of the enzyme when interacting with substrates.
Evolutionary changes that the amino acid sequences can undergo while subjected to the cold
include an increase in polar residues, glycines, and polar surface loops. Decreases include
hydrogen bonds, arginine to lysine ratio, prolines, and hydrophobic intermolecular forces among
subunits. These changes define a psychrophilic enzyme, or an enzyme that has adapted to colder
climates.
Amino acid sequence alignments are important tools for identifying the residue
modifications that have occurred in the same proteins. These residue changes are a direct
indicator of evolutionary changes that occur in proteins over time. In this lab, the primary
structures of four enzyme are studied: three from several barracuda species, and one from a
dogfish shark. Sequence aligntment tools such as the Clustal Omega tool allow scientists and
researchers to study evolution through biochemical and genetic means, and even enable the
determination of common ancestry among organisms.
MATERIALS AND METHODS
Obtained from the lab instructor via Blackboard, the lecture handout, lecture two PowerPoint,
DeepView Tutorial link, TA DeepView Tutorial PowerPoint, and literature article were all
referenced during the course of this lab. The 4.10 version of SPDBV for PC was used on a

Windows OS. Amino acid sequences were obtained from NCBI and Expasy. They were
sequenced with both the UniprotKB and Clustal Omega alignment tools. Three .pdb files, or 3-D
protein structures were downloaded from the NCBI website: 1LDN, 3LDH, and 6LDH. These
proteins were analyzed using DeepView by observing their amino acid active-sites, tetramer
structures, and amino acid substitutions. No immediate deviations were made from the protocol.
RESULTS
Part I-A.
For Part I-A, amino acid sequences for three species of barracuda and one species of dogfish
shark were collected from NCBI. These were found by searching lactate dehydrogenase A
Sphyraena and selecting entries AAB38888, AAB38887, and AAB38886. Lactate
dehydrogenase A Squalus was also then searched, wherein AAA91038 was selected.
Upon searching lactate dehydrogenase, 147922 hits appeared, indicative of the enormous
presence of LDH in a diversity of organisms.
Upon searching lactate dehydrogenase A Sphyraena, seven hits returned. They were not all for
the same protein, as the result details below each link varied with 332 aa and 377 aa.
AAB38888
1
61
121
181
241
301

mstkeklidh
evmdlqhggl
ipnivkyspn
hpsschgwiv
viklkgytsw
tdvihmtlkp

vmkeepigsr
flkthkivgd
cilmvvsnpv
gehgdssvpv
aigmsvadlv
eeekqlvksa

nkvtvvgvgm
kdysvtansr
diltyvawkl
wsgvnvagvs
esivknlhkv
etlwgvqkel

vgmasavsil
vvvvtagarq
sgfprhrvig
lqtlnpkmga
hpvstlvkgm
tl

lkdlcdelal
qegesrlnlv
sgtnldsarf
egdtenwkav
hgvkdevfls

vdvmedklkg
qrnvnifkfi
rhimgeklhl
hkmvvdgaye
vpcvlgnsgl

vmkeepigsr
flkthkivgd
cilmvvsnpv
gehgdssvpv
aigmsvadlv
eeekqlvksa

nkvtvvgvgm
kdysvtansr
diltyvawkl
wsgvnvagvs
esivknlhkv
etlwgvqkel

vgmasavsil
vvvvtagarq
sgfprhrvig
lqtlnpkmga
hpvstlvkgm
tl

lkdlcdelal
qegesrlnlv
sgtnldsarf
egdtenwkav
hgvkdevfls

vdvmedklkg
qrnvnifkfi
rhimgeklhl
hkmvvdgaye
vpcvlgnsgl

vmkeepigsr
flkthkivad
cilmvvsnpv
gehgdssvpv
aigmsvadlv
eeekqlvksa

nkvtvvgvgm
kdysvtansr
diltyvawkl
wsgvnvagvs
esivknctkc
etlwgvqkel

vgmasavsil
vvvvtagarq
sgfprhrvig
lqtlnpkmga
tqcprwsrgm
tl

lkdlcdelal
qegesrlnlv
sgtnldsarf
egdsenwkav
hgvkdevfls

vdvmedklkg
qrnvnifkfi
rhimgeklhl
hkmvvdgaye
vpcvlgnsgl

avgmacaisi
klvvitagar
lsglpmhrii
slkelhpelg

lmkdladeva
qqegesrlnl
gsgcnldsar
tdkdkenwkk

lvdvmedklk
vqrnvnifkf
frylmgerlg
lhkdvvdsay

AAB38887
1
61
121
181
241
301

mstkeklinh
evmdlqhggl
ipnivkyspn
hpsschgwiv
viklkgytsw
tdvihmtlkp

AAB38886
1
61
121
181
241
301

mstkekligh
eamdlqhgsl
ipnivkyspn
hpsschgwiv
viklkgytsw
tdvihmtlkp

lactate dehydrogenase A Squalus: AAA91038

1
61
121
181

matlkdklig
gemmdlqhgs
iipdivkhsp
vhssschgwv

hlatsqeprs
lflhtakivs
dciilvvsnp
igehgdssvp

ynkitvvgvg
gkdysvsags
vdvltyvawk
vwsgmnvagv

241 eviklkgyts waiglsvadl aetimknlcr vhpvstmvkd fygikndvfl slpcvldnhg

301 isnivkmklk pdeeqqlqks attlwdiqkd lkf

Figure 1. Amino acid sequences for three species of barracuda and one species of dogfish shark,
obtained from NCBI.
Part I-B.
In order to find the same sequences of the four proteins searched in NCBI, the same three-letter
and five-number combinations were entered directly into the search engine for Expasy. An
abundance of information was gathered from each entry page for the corresponding enzymes:
protein, gene, organism, function (catalytic activity, pathway: pyruvate fermentation to lactate,
binding sites), names and taxonomy, subcellular location, PTM/processing, interaction, structure,
family & domains, sequence, cross-references, entry information, similar proteins.
A sequence alignment was performed on the FASTA sequences using the UniProtKB tool.
Subsequently, a sequence alignment was performed on these same FASTA sequences using the
Clustal Omega tool from the European Bioinformatics Institute.

Figure 2. Protein sequences of LDHA from UniProtKB alignment tool.

Figure 3. Protein sequence alignment created with Clustal Omega tool.

The UniProt tool highlights the amino acids based off of annotation and properties. This is
especially useful for pinpointing information such as the active site, chains, helices, and binding
sites. It also distinguishes amino acids based off their characteristics, including charge,
aromaticity, and hydrophobicity. Both provide a phylogenetic tree. The Clustal Omega tool
displays colors. With color sequences, it is easy to report patterns in the enzyme sequences.
Part II.
In Part II, three 3D protein structures are downloaded from NCBI. From lactate dehydrogenase
Squalus, 3LDH and 6LDH were downloaded, where 6LDH is the dogfish shark without
substrates and cofactors. From lactate dehydrogenase Bacillus stearothermophilus, 1LDN was
downloaded.

Figure 4. Protein Sequence alignment made with UnitProtKB aligntment tool for 1LDN, 3LDH,
and 6LDH.

Figure 5. Protein sequence of 1LDN, 3LDH, and 6LDH with Clustal Omega Tool.

Part III-A
In DeepView, the first 3D protein structure analyzed is 1LDN, derived from the thermophile.
The structure is modified to expose a colored ribbon depiction of the two tetramers. The H chain
is then isolated while pyruvate and NADH are displayed. Arginine amino acids were displayed
to isolate the active-site molecule in relation to pyruvate.

Figure 6. The two tetramers of 1LDN. In the protein, there are two tetramers displayed, with
each monomer highlighted by a different ribbon color: yellow, green, red, blue, light blue, white,
magenta, and pink. The two tetramers appear like two separated globular proteins, and they are
not the same.

Figure 7. Chain H of 1LDN with pyruvate in blue stick and ball structure and NADH in yellow
stick and ball structure.

Figure 8. Chain H with pyruvate, NADH and arginine molecules. Nitrogen sticks are depicted in
blue sticks, while oxygen atoms are depicted with red sticks.

Figure 9. Distance tool calculation yielding 4.34 Angstroms between pyruvate and active-site
Arginine-169. In the picture to the left, the Nitrogen molecules are indicated by the blue ends of
the stick-model. In the picture to the right, the pyruvate bonds are highlighted red to indicate the
oxygen molecules.

Figure 10. Distance tool identifying the active-site Arginine as number 169.

Figure 11. Zoomed-out view of the Arginine-169, pyruvate, and NADH in chain H amongst
other arginine molecules.

Figure 12. Chains E, F, G, and H forming a tetramer. The pyruvate molecule can be seen at the
interface of all four chains towards the surface of the molecule.

Figure 13. Zoomed-in view of the pyruvate and Arginine-169 active-site on the tetramer
interface.
Using the control panel, the arginine amino acids were displayed as stick structures for chain H
by checking off the first two columns. The correct orientation occurs when the carbon
connecting the two amine groups is open to a bent end of a pyruvate molecule with two oxygen
atoms. The active-site arginine was determined by the orientation of the pyruvate end with two
oxygen atoms with the nearest arginine molecule side chain. The distance was measured between
the center of the pyruvate molecule and the closest arginine carbon. The shortest distance that
was measured was 4.34 Angstroms from the Arginine-169 amino acid. It must be noted that this
distance was measured between two carbon atoms.
The active site for the pyruvate in relation to Arginine-169, when chains E, F, and G are also
displayed alongside H, can be seen at the planes of the four monomers, or where they meet.
This can be seen in Figure 12 and 13. The active site is more towards the surface of the overall
molecule, rather than being deeply internal.
Part III-B
The 3D protein structure of 3LDH is analyzed by observing its secondary helical and sheet
structures as well as locating the active-site of histidine and arginine.

Figure 15. 3LDH molecule with strands in yellow, and the helices in yellow.

Figure 16. The active-site of Arginine-171 in relation to pyruvate. The distance between carbons
is 4.37 Angstroms. The red sticks are oxygen atoms, and the blue sticks are nitrogen atoms.

Figure 17. The active-site for Histidine-195 in relation to pyruvate. The distance is 2.96
Angstroms between carbon atoms. The red bonds represent the oxygen molecules of pyruvate,
and the blue bonds represent the nitrogen bonds of histidine.
There are twelve helices and nine sheets. The amino acids that play a part in the active-site for
pyruvate are Arginine-171 and Histidine-195. These amino acids were identified by measuring
their distances from pyruvate, and their results were 4.37 and 2.96 Angstroms from carbon
atoms, respectively.
Part III-C.
3LDH and 6LDH were imported into the DeepView program to be displayed simultaneously.
The 6LDH represented the lactic dehydrogenase from the dogfish shark without a substrate and
cofactor by excluding pyruvate and NADH. The two enzymes were colored differently by
ribbon, where 3LDH was pink and 6LDH was yellow.

Figure 18. The merged 3LDH and 6LDH, where 3LDH is colored pink and 6LDH is colored
yellow.

Figure 19. A depiction of the changed loop, where the pink and yellow strands divert.

Figure 20. Display of the pyruvate in relation to the loop, where the pyruvate molecule is to the
right and the loop to the left.

Figure 21. The Control View window displaying the amino acids that have shifted and remained
the same for 3LDH. The amino acids in red texts have remained the same, while the black ones
have shifted.

Figure 22. The Control View window displaying the amino acids that have shifted and remained
the same for 6LDH. The amino acids in red texts have remained the same, while the black ones
have shifted.
The RMS deviation of the two fit proteins is 1.3 Angstroms.
DISCUSSION/CONCLUSIONS
Parts I & II.

Figure 23. A marked sequence alignment, where the black marks at positions 8, 61, 68, and 223
indicate changes in amino acids of barracuda when compared vertically.
When enzymes are subjected to colder temperature, they must substitute their residues that
would ordinarily be rigid for residues that promote flexibility. This allows the kinetic properties
of the enzyme to stay the same, as seen in Km, thus resulting in an increased efficiency.
In psychrophilic enzymes, the electrostatic repulsions between amino acids should increase while
the attractions decrease in order to promote overall protein flexibility when exposed to colder
environments. This allows the spaces between molecules to increase, lessening the compactness
of the overall enzyme and removing rigidity.
The internal structure of a protein usually features hydrophobic amino acids. In psychrophilic
enzymes, the hydrophobic interactions and residues are expected to decrease. In addition,
proteins tend to be made of a hydrophobic core and a hydrophilic surface to protect the
hydrophobic core from exposure to immiscible solvents. Because flexibility can be improved
when solvent interactions are induced with residues on the outermost part of a protein,
hydrophilic amino acids are more likely to replace hydrophobic ones in this location.
There are several variations in amino acid sequences of the barracuda. These occur at position 8,
61, 68, and 223, as discussed by Holland et al in reference 2.
The shifts are as follows:
Position 8: aspartic acid, arginine, glycine
Position 61: valine to alanine
Position 68: glycine to serine

Position 223: threonine to serine

Figure 24. A comparison of the flexibility of amino acids, where glycine is the most flexible and
proline is the least.6
According to Figure 24, the transition of aspartic acid to arginine to glycine indicates increased
flexibility across the three species of barracuda. Additionally, the transition from valine to
alanine also demonstrates a jump in increased flexibility. While glycine to serine does not
support this trend, it still represents an increase in polar amino acids, thus supporting the
psychrophilic modifications of greater polar residues.6
According to Holland et. al, In position 8, the barracuda that habitats the North, SPHAG, has a
modified glycine, and a removed aspartic acid. This can indicate increased thermal stability, as it
is likely subjected to colder water temperatures.
When comparing the three sequences of barracuda with the dogfish shark sequence, there are
even more positions of sequence variations. The Clustal Omega Tool FAQ provided insight on
the symbols that accompany each position: asterisks indicate positions with unchanged residue,
colons indicate changes in residue that retained strongly similar properties, and periods represent
residue modification in which the similarities between properties are weak. While most of the
symbols consist of asterisks and colons, there are periods at a series of positions. These periods,
meaning strong residue variation, are the best indicator of evolutionary changes in the overall
enzyme. The periods almost always feature two different colors for the residue changes, while
the colon either retains one overall color or displays two. These color changes are direct
indicators of amino acid properties: red represents small and hydrophobic, blue represents acidic,
magenta represents basic, green represents hydroxyls, sulfhydryls, and amines. A period
accompanied by a color change represents the strongest diversion of residues in a sequence
alignment.
Part III
The active-site amino acids can be determined by the distance and orientation of the substrate
and amino acid. In part A and part B, the arginines that are classified as being active-site are the
least distance away from the substrate, pyruvate, whilst being oriented properly towards it. The
acid side of the pyruvate with the two oxygens attached to one carbon are facing the end of the
amino acid chain. The oxygens are in red, while the end of the arginine chain is in blue,
indicating a charged nitrogen. This also applies to the histidine in part B. These atoms facing one
another ensures an orientation that stabilizes the substrate, reducing charge. The decreased
distance allows this to happen.
The loop in Part C indicates where the secondary structure of the enzyme turns, enabling folding.
This promotes binding of substrate and overall enzyme. The reason why this loop can be
identified is because 3LDH includes the substrate, pyruvate, and the cofactor, NADH, while
6LDH represents this enzyme without them. Therefore, when DeepView displays both of these
enzymes simultaneously the structural differences related to binding can be identified.

REFERENCES
1. Some Like it Cold. Lab Handout. 2016. John R. Engen.
2. Evolution of Lactate Dehydrogenase-A Homologs of Barracuda Fishes (Genus Sphyraena)
from Different Thermal Environments: Differences in Kinetic Properties and Thermal Stability
Are Due to Amino Acid Substitutions Outside the Active Site. Biochemistry. 36: 3207-3215.
1997. Linda Z. Holland et. al.
3. Tutorial for DeepView Swiss-PdbViewer. Academic Press. 3rd Edition. 2006. Gale Rhodes.
4. Quick Introduction to DeepView. Lab Handout. 2016. John R. Engen.
5. Lab 2 Lecture. Lab Handout. 2016. John R. Engen.
6. A Conformational Flexibility Scale for Amino Acids in Peptides. Chemie International
Edition. 42. 2002. Feng Huang, Werner M. Nau.