Topic 6 - Enzymes

Question 1

Spontaneous Reactions

Answer 1

• all are thermodynamically or energetically favorable
• all reactions of enzymes are spontaneous
• enzyme does not change free energy of substrate of product
• enzyme does not affect whether rxn is thermodynamically favorable, will merely catalyze it

Question 2

Properties of Enzymes

Answer 2

1. Catalysts: Not used up in the reaction (Regenerated)
2. Biological catalysts have higher reaction rates than chemical catalysts
3. Mild reaction conditions: normal pH & temperature, occur within biologically appropriate conditions
4. Greater rxn specificity: an enzyme can be very specific for its substrate (glucokinase can only phosphorylate glucose) or not very specific (hexokinase can phosphorylate glucose, frucotose, and mannose)
5. Capacity for regulation: enzyme activity can be increased/decreased via covalent modification (irreversible or reversible) or non-covalent modification (allosteric/regulatory enzymes)

Question 3

Enzyme Classes

(6)

Answer 3

1. Oxidoreductases
2. Transferases
3. Isomerases
4. Hydrolases
5. Lyases
6. Ligases

Question 4

Answer 4

1. Oxidoreductase: The transfer of electrons

• Catalyze redox reactions - thus requires an electron acceptor and electron donor
• Ex:
• COMMON NAME: lactate dehydrogenase
• SYSTEMATIC NAME: electron donor:acceptor + oxidoreductase - lactate:NAD+ oxidoreductase
• electrons from lactate are transferred to NAD+ (as hydride ion, a hydrogen with an extra electron) –> NAD+ becomes NADH.
• Reduction = loss of hydrogens. (NOTE: it is NOT protonation b/c NAD is not picking up a proton, it is picking up a hydride ion).

Question 5

Answer 5

2. Transferase: Transfer of functional groups from one molecule to another: involves 2 molecules and a functional group such as an amino group of phosphyl group. It is NOT just the addition of a functional group.

• Subgroups: Kinases - transfer phosphate groups from ATP to substrate
• Ex:
• COMMON NAME: phosphofructokinase (PFK)
• SYSTEMATIC NAME: molecule + kinase - fructose-6-phosphate kinase
• phosphofructokinase transfers a phosphate from ATP to the substrate

Question 6

Answer 6

3. Isomerase: INTRAmolecular rearrangement

• Ex:
• COMMON NAME: triose phosphate isomerase
• SYSTEMATIC NAME: substrate + isomerase - dihydroxyacetonephosphate isomerase

Question 7

Answer 7

4. Hydrolase: Single bond cleavage via addition of H2O OR bond formation via the removal of H2O

• Ex:
• SYSTEMATIC NAME: compound to be cleaved + hydrolase - peptide hydrolase (peptidase) - break peptide bond by adding water, make peptide bond by removing water
• Note: peptide bond = bond between carbonyl group and amino group of aa

Question 8

Answer 8

5. Lyase: Group elimination to form a double bond

• NOTE: POE - Eliminate the other options first, because this is usually difficult to identify !
• NOTE: May be confused with hydrolase, but there is no conversion to double bond like with lyase
• Ex:
• COMMON NAME: enolase
• SYSTEMATIC NAME: 2-phosphoglycerate lyase

Question 9

Answer 9

6. Ligase: bond formation coupled to ATP hydrolysis

• NOTE: do not confuse with transferases
• Ex:
• COMMON NAME: pyruvate carboxylase
• SYSTEMATIC NAME: molecule:molecule + ligase - pyruvate:carbon dioxide ligase

Question 10

Enzymes & Transition State

Answer 10

• determines rate of reaction
• magnitude of change in free energy/Ea (activation energy)
• enyzmes decrease Ea by stabilizing the transition state
• BAD: if enzyme binds to substrate perfectly and interacts with it - results in a very stable enzyme-substrate complex - lowers energy and increases Ea
• NEED: enzyme that perfectly binds the transition state; interactions that stabilize TS lower the energy of TS –> decreases Ea
• catalysts: stabilize TS –> lower energy of TS
• enzyme catalysis: 2 step process - substrate must bind to enzyme & E-S complex catalyzes conversion

Question 11

Enzymes: Binding Sites

(Properties)

Answer 11

• Active Site: Substrate Binding Site + Catalytic Site
• Regulatory Site: a second binding site for other molecules other than the substrate; it is separate from the active site. Binding by regulatory molecule affects the active site - alters the efficiency of catalysis, and/or improves or inhibits catalysis
• Both active & regulatory site should be complementary to the ligand that they are trying to bind
• Three dimensional space; Occupies small part of enzyme volume; Clefts or crevices - where the substrate binds
• Ligands (substrate or effector) are bound by multiple weak interactions
• Specificity from precise arrangement of atoms in active site: correct orientation and complementarity to size, shape, and charge/polarity to favorably bind the ligand
• Geometric (physical) complementarity
• Electronic (chemical) complementarity
• Charge and polarity

Question 12

Enzyme-Substrate Complex

Answer 12

• active site of enzyme will have same shape as substrate: same size and shape, as well as complementarity, such as +charge to interact with - charges, electron acceptor interacting with electron donor, etc.

Question 13

Stereospecificity leads to Substrate specificity

Answer 13

Explanation for the specificity of the enzyme to its substrate

Explanation for why citrate always becomes R-isocitrate: because of specificity of substrate binding. Only 3 out of the 4 attachments can interact with the enzyme, and will interact in a specific way/orientation to always give R-isocitrate.

Enzymes vary in geometric specificity: Alcohol dehydrogenase can catalyze the oxidation of 3 different alcohols, but prefers ethanol > methanol > isopropanol. Ethanol fits best, methanol is smaller but can still react with the pocket, and isopropanol is bigger but can still react.

Question 14

Trypsin

Answer 14

cleaves after long +charged side chains: has a long groove with a negative charge to stabilize +charge of aa

Question 15

Chymotrypsin

Answer 15

has large, hydrophobic pocket to accommodate rings of large, aromatic side chains

Question 16

How do we identify and characterize active sites?

Answer 16

• Using model substrates (which are similar to the desired substrate) and competitive inhibitors to determine structure (size, shape, charges) of active sites.
• Can therefore identify amino acids of enzymes that are involved in binding & catalysis

Question 17

Model Substrate: Chymotrypsin - What is required for binding & catalysis?

Answer 17

Model substrates will be similar to the actual substrate, but different in a few regions to assess the importance of these regions in the enzyme - will the enzyme still function after these regions have been changed?

1. Do we need the whole peptide chain? NO: the amino acid can still be modified at the N or C-terminus, or have an additional amino group, and still be considered a good substrate b/c chymotrypsin can still cleave the peptide bond
2. Do we need the a-amino group? NO: when a-amino group is gone, the peptide is still be a good substrate. Only alpha-Carbon, carbonyl, and peptide is required for binding & catalysis. The amino group at the end is not required either, as long as it is an electronegative atom, chymotrypsin can still cleave the bond
3. Does R group have to be Phenyalanine, Tyrosine, or Tryptophan? NO: as long as R group is relatively bulky and generally hydrophobic, it will bind to the pocket

Conclusion: Chymotrypsin recognizes a bulky hydrophobic group attached to a “peptide bond” (a carbonyl attached to an electronegative atom) - this is all that is required for binding

Question 18

Competitive Inhibitors: Arginase

Answer 18

Competitive Inhibitors: Compounds that compete with the substrate for binding; they bind in the active site but enzymes cannot use them as substrates - they inhibit catalysis

Assay to see if it is an inhibitor: run an enzyme assay with normal substrate & include inhibitor –> is there decreased activity?

Arginine is cleaved into ornithine & urea; Arginase will bind to compounds that look similar to arginine –> must have 3 charges and a relatively long chain in between the alpha-carbon and the charge at the end of the R-group