C15 1 Hexokinase

Chapter 15: Bioinformatics Exercise 15-1
Hexokinase Structure and Ligand Binding
Exercise Goals
 Understand structure–function relationships in hexokinase.
 Explore the active site and allosteric binding site of hexokinase.
Difficulty
 17 questions
 Computer-Related: Difficult
 Subject-Related: Difficult
Resources & Websites

 Figure 15-2, Chapter 15
 Bioinformatics Exercises 6-1 to 6-5, 7-1, 7-2, 11-1, 11-3, and 13-1
 Protein Data Bank:
o http://www.rcsb.org
 Sequence and Structure Alignment Tool:
o http://pdb.org/pdb/home/home.do#Category-analyze
 Protein Workshop Tutorial:
o http://www.rcsb.org/pdb/staticHelp.do?p=help/viewers/proteinWorkshop_viewer.html
 Ligand Explorer Tutorial:
o http://www.rcsb.org/pdb/staticHelp.do?p=help/viewers/ligandExplorer_viewer.html
Exercise
In glycolysis, the phosphorylation of glucose by hexokinase serves as the entry point for
the pathway. Accordingly, this exergonic reaction is highly regulated through allosteric
and competitive inhibition, such that binding of product (glucose-6-phosphate) or the
cosubstrate (ATP with no bound Mg2+) decreases the enzymatic rate. Hexokinase is also a
model enzyme for studying induced fit, where conformational changes associated with
substrate binding are essential for catalytic activity. In this exercise, the structures of
different hexokinases will be studied using Protein Workshop, Ligand Explorer, and the
Sequence and Structure alignment tool from the Protein Data Bank.
Instructions
1. Go to www.rcsb.org.
2. Sequentially search for these PDB entries:
a. 3B8A; review the information provided on the Summary, Sequence, and
Annotations tabs.
b. 1IG8
c. 1BG3
3. View the structures using Protein Workshop, as described in Bioinformatics
Exercises 5-1, 6-1 to 6-5, 11-1, and 11-3.
This study source was downloaded by 100000867365404 from CourseHero.com on 06-08-2023 22:03:31 GMT -05:00
https://www.coursehero.com/file/51786978/C151Hexokinasedocx/
4. View the ligand binding sites using Ligand Explorer, as described in Bioinformatics
Exercise 7-1.
5. To compare 3B8A and 1IG8, use the Sequence and Structure Alignment tool, as
described in Bioinformatics Exercise 13-1.
Assessment Questions
1. According to the PDB entry for 3B8A, hexokinase is a two-domain protein, where the
nucleotidyltransferase domain is characterized as an ________.
A. alpha-helical protein with a 3-layer sandwich architecture

B. alpha-helical protein with a 2-layer sandwich architecture
C. alpha/beta protein with a 3-layer sandwich architecture
D. alpha/beta protein with a 2-layer sandwich architecture
2. As indicated by the information on the Sequence tab for PDB entry 3B8A, residues
116–120 comprise which stable structure?
A. alpha helix
B. beta sheet
C. turn
D. 3/10 helix
3. Use Protein Workshop to choose the statement that best describes the glucose
binding site on hexokinase (3B8A).
A. The glucose binding site is in a deeply buried cleft at the interface of the
enzyme’s domains.
B. The glucose binding site resides on the surface of the protein at the interface
of the enzyme’s domains.
C. The glucose binding site is in a deeply buried cleft that can be accessed from
either side of the protein.
D. The glucose binding site is in a deeply buried cleft that is mostly hydrophobic.
4. The majority of amino acids that comprise the glucose binding site in hexokinase
(3B8A) are found on _______ within the protein.
A. alpha helices
B. beta sheets
C. disordered regions
D. loop regions
5. According to the structure for 3B8A, how does the glucose C6 hydroxyl group
interact noncovalently with the protein?
A. only as a hydrogen bond donor

B. only as a hydrogen bond acceptor
C. as both a hydrogen bond acceptor and donor
D. through hydrophobic interactions
6. Consistent with the structure for 3B8A, the glucose hydroxyl group that contains the
atom “O4 Residue BGC 501” serves as a hydrogen bond _____ for ___ different
hydrogen bonds.
A. donor; 1
B. donor; 3
C. acceptor; 1
D. acceptor; 3
7. According to the structure for 3B8A, which oxygen atom of the glucose ligand is NOT
involved in a hydrogen bond?
A. the oxygen of C2
B. the oxygen of C3
C. the oxygen of C4
D. the oxygen of C5
8. According to the structure for 3B8A, which carbon atoms of the glucose ligand are
involved in hydrophobic interactions with the protein?
A. C1, C2, C3, and C4

B. C5 and C6
C. C4, C5, and C6
D. C1, C4, C5, and C6
9. Analysis of the noncovalent interactions in 3B8A indicates that hydrogen bonds have
a length of ______, while hydrophobic interactions have a length of _____.
A. 3.7–3.9 Å; 2.6–3.2 Å
B. 2.6–3.2 Å; 3.7–3.9 Å
10. As observed in Protein Workshop for PDB entry 1BG3, the rat brain hexokinase 1 is a
dimer that forms under which conditions, according to the PDB entry page?
A. The protein dimer is the native form.

B. The protein dimerizes as the result of genetic engineering.
C. The protein dimer forms at high concentrations of the protein, but only in the
absence of the inhibitor.
D. The protein dimer forms at high concentrations of the protein, especially in
the presence of the inhibitor.
11. As observed in the structure for 1BG3, the substrate and inhibitor binding sites are
located ______ of the monomeric unit of the enzyme, and the anomeric carbons of
the two ligands are separated by ______ angstroms.
A. on opposites sides; 46.4

B. on opposites sides; 7.3
C. in the same cleft region; 11.7
D. in the same cleft region; 7.3
12. According to the surface rendering of 1BG3, the binding of glucose-6-phosphate to

the allosteric site inhibits catalysis by preventing ATP from binding to the active site.
A. True
B. False
13. In the structure of 1BG3, which amino acids are hydrogen bonded to both glucose
and glucose-6-phosphate?
A. Thr 680
B. Asp 657
C. Asp 656
D. No amino acids are hydrogen bonded to both compounds.
14. Which statement is consistent with the sequence and structural alignment of PDB
entries 1BG3 and 3B8A (using the Sequence and Structure alignment tool)?
A. These proteins do not possess identical primary structures, yet their tertiary
structures are very similar.
B. These proteins possess identical primary structures, and their tertiary
structures are very similar.
C. These proteins do not possess identical primary structures, and their tertiary
structures are not very similar.
D. These proteins possess identical primary structures, yet their tertiary
structures are not very similar.
15. Which statement regarding the impact of glucose binding is consistent with the
comparison of PDB entries 1BG3 and 3B8A? [For this question use the following
rendering options: View each protein in Protein Workshop (in separate windows);
turn on the Ribbon for both proteins; turn on the Atom & Bonds for residues Lys 176
and Tyr 308, and display these amino acids in CPK style.]
A. These proteins cannot be compared since they represent two distinct
isozymes of hexokinase and have unique tertiary structures.
B. These renderings indicate that there are no conformational changes upon
glucose binding.
C. These renderings indicate that only the side chains of Lys 176 and Tyr 308
move closer together upon glucose binding.
D. These renderings indicate that the two domains of hexokinase move closer
together upon glucose binding.
16. In the structural alignment of PDB entries 1BG3 and 3B8A, does Lys 176 or Tyr 308
experience a larger change in conformation (backbone and side chain) upon glucose
binding? [To answer this question, use the Sequence and Structure alignment tool,
select all tyrosine residues, view all tyrosine residues by selecting the “Bond” option
and choosing 0.20 A, then repeat these steps for all lysine residues. Rotate the
structure until the cleft and residues Lys 176 and Tyr 308 are clearly visible (as in
Question 15).]
A. The backbone of Lys 176 experiences a larger change in conformation.
B. The backbone of Tyr 308 experiences a larger change in conformation.
C. The backbones of both amino acids experience the same degree of
conformational change.
D. Neither amino acid experiences a conformational change upon glucose
binding.
17. The information presented in Questions 14–16 is consistent with which type of
ligand binding model for glucose and hexokinase?
A. The lock and key model, since no major conformational changes are
observed.
B. The induced fit model, since changes only in side chain orientation are
observed.
C. The lock and key model, since significant changes in the backbone
conformation are observed.
D. The induced fit model, since significant changes in the backbone
conformation are observed.
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Chapter 15: Bioinformatics Exercise 15-1

Hexokinase Structure and Ligand Binding

Resources & Websites

A. alpha-helical protein with a 3-layer sandwich architecture

A. only as a hydrogen bond donor

A. C1, C2, C3, and C4

A. The protein dimer is the native form.

A. on opposites sides; 46.4

12. According to the surface rendering of 1BG3, the binding of glucose-6-phosphate to

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