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)
1. Oxidoreductase: The transfer of electrons
- Catalyze redox reactions - thus requires an electron acceptor and electron donor
- 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
- 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
- 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
- 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
- COMMON NAME: enolase
- SYSTEMATIC NAME: 2-phosphoglycerate lyase
6. Ligase: bond formation coupled to ATP hydrolysis
- NOTE: do not confuse with transferases
- 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
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
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.
cleaves after long +charged side chains: has a long groove with a negative charge to stabilize +charge of aa
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?
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
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