Chapter 3: Enzyme Kinetics Flashcards
Enzymes (E)
- Are biological catalysts
- Are proteins (few are RNA: ribozymes)
- Increase reaction rate by lowering activation energy (biggest energy barrier)
- All enzymatic reactions are reversible, enzymes have a high degree of specificity (binds to closely related substrates) and are very large
- Unaltered in reactions & Can’t change the ground state energy levels of reactants or products
Reaction diagram for S->P
- Ground state substrate contorts until it becomes the transition state (TS) for a very very short time
1a. Transition state has an equal chance to become S or P - Difference in energy between substrate and transition state is the activation energy
2a. Step with highest hill=activation energy=rate limiting/determining step (what determines speed of reaction)
Enzyme catalyzed reaction scheme
- E+S->ES->EP->E+P
- Binding energy is the difference between the activation energy (highest hill) of the uncatalyzed reaction versus the catalyzed reaction
ES complex formation theories
- Lock and Key: Older theory that says that active site matches substrate like a lock matches a key
- Induced fit theory:
2a. Says the active site of an enzyme matches the transition state
2b. Both enzyme and substrate change shape to first form the ES complex and again in the transition state
Binding energy
- Difference in energy between the transition states of enzyme catalyzed reactions and no enzyme reactions
- Formed by weak and covalent forces
2a. Weak (most important) forces: allow for an induced fit of the enzyme into the substrate
Cofactors
- Aid enzymes to reach their optimal activity
- Can be organic or inorganic and may bind tight or loosely
2a. Organic cofactors: coenzymes ->may be vitamins or vitamin derivatives (Ex: AMP)
2b. Inorganic cofactors: metal ions - Tightly binding cofactors are prosthetic groups that covalently bind to their enzyme (ex: heme to hemoglobin)
What factors affect enzymatic reactions?
- Temperature: increase in temp=increase in rate (more collisions)=too much denatures enzyme=denatured enzyme decreases rate
1a. Optimal temp=37C - PH: optimal pH is up to enzyme
- Reactant concentration: increase substrate (reactant)=increase rate =too much saturation decreases rate
Dissociation constant (Kd)
- Kd=([E][S])/[ES]
1a. Low Kd=high ES affinity (ES prefer being bound rather than not)
1b. If Kd is high=low binding rate
Michaelis-Menten equation
- In E+S (k-1)<—> (k1)ES (k2)—->P+E
1a. Vo=((kcat)([E]total)([S])/(Km+[S])
1b. Vmax=kcat*[E]total
1c. Catalytic efficiency=kcat/Km - If [S]=Km : Vo=Vmax/2
- Variables:
3a. Vo: Instantaneous rate that the product is formed at the beginning of the reaction
3b. Michaelis constant (Km): Being half equal to the concentration of substrate required to make an initial reaction run at Half its maximum rate (Km=(k-1 + k2)/k1)
3c. Vmax: Maximum initial rate possible / when there is so much substrate that every enzyme has one (enzyme is saturated)
3d. Kcat: turnover constant in (1/s)
3e. higher Km=lower affinity
Specificity constant
- Specificity constant=kcat/Km
1a. Catalytic efficiency of ES Pair: higher specificity=E is more efficient at converting S to P - Max # this could be is 10^8 and 10^9 1/Ms
Reversible inhibition: competitive inhibitors
- Bind active site because they resemble substrate, can be overcome if high [S]
1a. Raise Km (new Km is apparent Km), don’t effect Vmax
Reversible inhibition: uncompetitive inhibitors
- Bind to ES complex (don’t resemble substrate)
1a. Lower Km, lower Vmax (new Vmax is apparent Vmax) - They bind to E after substrate is bound
- Work better if substrate and inhibitor concentrations are increased
Reversible inhibition: mixed inhibitors
- Bind to either ES complex or free E (don’t resemble substrate)
1a. Lower or unchanged Km, lower Vmax - Noncompetative inhibitor: a type of mixed inhibitor that binds to free E equally and as often as it binds to ES
2a. Unchanged Km, lowers Vmax
Michaelis menten equation + reversible inhibitor
- Vo=((Vmax)([S])/((aKm) + (a’[S]))
1a. Apparent Km=aKm/a’
1b. Apparent Vmax=Vmax/a’ - a> or = 1 and a’ >or = 1
2a. Competative inhibiton: a’=1
2b. Uncompetitive inhibition: a= 1
Lineweaver-Burk Plots/double reciprocal plot
- The reciprocal of michaelis menten equations:
1a. 1/Vo=Km/((Vmax[S]) + 1/Vmax) - In plot
2a. X intercept: -1/Km (if increased=Km increases)
2b. Y intercept: 1/Vmax (if decreased=Vmax increases)
2c. M=Km/Vmax (if decrease=more efficient)
Irreversible inhibitors
- Form one or more stable bonds (covalent or not) with an enzyme which interrupts the normal function of the enzyme
- Tend to be highly toxic
Types of feedbacks from enzymes
- Negative feedback/feedback inhibition: occurs when the product downstream inhibits the upstream reaction
- Positive feedback: series of enzymatic reactions that stimulate the enzymes upstream (Ex: oxytocin in birth)
Regulation by enzymes: allosteric effectors/modulators
- Small metabolites or cofactors that reversibly / noncovalently bind to allosteric enzymes at the regulator site which cause them to change their shape (these effectors can be inhibitory or stimulatory)
- Show sigmoidal saturation curve from cooperativity
Regulation by enzymes: proteolytic cleavage (irreversible covalent modification)
- Proenzymes (ex: zygomens) are proteolytically cleaved to be active
Regulation by enzymes: control proteins
- Protein subunits that bind to certain enzymes to activate or inhibit them
- Ex: GPCR
Regulation by enzymes: reversible covalent modification
- Regulatory mechanism that occurs in many ways
- Example: phosphorylation (PTM that occurs on T, S, Y -bc of their OH)
2a. Kinase: transfers phosphate (transferase) from organic molecules (ATP)
2b. Phosphatase: removes phosphate using water (hydrolase) which makes inorganic phosphate
2c. Phosphorylase: adds inorganic phosphate to a substrate (transferase) - Example: deamidation:
3a. Amide of asparagine or glutamine is cyclicized which loses a NH3
Enzyme classification
- Ligament: joins 2 molecules (ex: synthetase)
- Isomerase: moves functional groups from one position to another within a molecule (ex: mutase)
- Lyases: breaks substrates apart without water (ex: decarboxylase, aldolase, synthase)
- Hydrolase: hydrolysis (exergonic) to break down macromolecules with water (ex: phosphatase)
- Oxidoreductase: transfers electrons from reluctant to oxidant (ex: dehydrogenase (uses NAD+, NADP+, FAD) , oxidase)
- Transferase: moves functional groups from one molecule to another (ex: kinase, phosphorylase)
Cooperativity
- If H=1 : no cooperative
- If H>1: Cooperative
Independent vs dependant variable in science
- Independent: x axis, unchanged, cause etc (time)
- Dependant: y axis