Topic 6 - Enzymes Flashcards
Spontaneous Reactions
- 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
Properties of Enzymes
- Catalysts: Not used up in the reaction (Regenerated)
- Biological catalysts have higher reaction rates than chemical catalysts
- Mild reaction conditions: normal pH & temperature, occur within biologically appropriate conditions
- 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)
- Capacity for regulation: enzyme activity can be increased/decreased via covalent modification (irreversible or reversible) or non-covalent modification (allosteric/regulatory enzymes)
Enzyme Classes
(6)
- Oxidoreductases
- Transferases
- Isomerases
- Hydrolases
- Lyases
- Ligases
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).
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
3. Isomerase: INTRAmolecular rearrangement
- Ex:
- COMMON NAME: triose phosphate isomerase
- SYSTEMATIC NAME: substrate + isomerase - dihydroxyacetonephosphate isomerase
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
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
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
Enzymes & Transition State
- 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
Enzymes: Binding Sites
(Properties)
- 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
Enzyme-Substrate Complex
- 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.
Stereospecificity leads to Substrate specificity
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.
Trypsin
cleaves after long +charged side chains: has a long groove with a negative charge to stabilize +charge of aa
Chymotrypsin
has large, hydrophobic pocket to accommodate rings of large, aromatic side chains
How do we identify and characterize active sites?
- 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
Model Substrate: Chymotrypsin - What is required for binding & catalysis?
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?
- 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
- 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
- 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
Competitive Inhibitors: Arginase
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
