Chapter 3: Enzyme Kinetics

Question 1

Enzymes (E)

Answer 1

1. Are biological catalysts
2. Are proteins (few are RNA: ribozymes)
3. Increase reaction rate by lowering activation energy (biggest energy barrier)
4. All enzymatic reactions are reversible, enzymes have a high degree of specificity (binds to closely related substrates) and are very large
5. Unaltered in reactions & Can’t change the ground state energy levels of reactants or products

Question 2

Reaction diagram for S->P

Answer 2

1. 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
2. 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)

Question 3

Enzyme catalyzed reaction scheme

Answer 3

1. E+S->ES->EP->E+P
2. Binding energy is the difference between the activation energy (highest hill) of the uncatalyzed reaction versus the catalyzed reaction

Question 4

ES complex formation theories

Answer 4

1. Lock and Key: Older theory that says that active site matches substrate like a lock matches a key
2. 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

Question 5

Binding energy

Answer 5

1. Difference in energy between the transition states of enzyme catalyzed reactions and no enzyme reactions
2. Formed by weak and covalent forces
   2a. Weak (most important) forces: allow for an induced fit of the enzyme into the substrate

Question 6

Cofactors

Answer 6

1. Aid enzymes to reach their optimal activity
2. 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
3. Tightly binding cofactors are prosthetic groups that covalently bind to their enzyme (ex: heme to hemoglobin)

Question 7

What factors affect enzymatic reactions?

Answer 7

1. Temperature: increase in temp=increase in rate (more collisions)=too much denatures enzyme=denatured enzyme decreases rate
   1a. Optimal temp=37C
2. PH: optimal pH is up to enzyme
3. Reactant concentration: increase substrate (reactant)=increase rate =too much saturation decreases rate

Question 8

Dissociation constant (Kd)

Answer 8

1. 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

Question 9

Michaelis-Menten equation

Answer 9

1. 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
2. If [S]=Km : Vo=Vmax/2
3. 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

Question 10

Specificity constant

Answer 10

1. Specificity constant=kcat/Km
   1a. Catalytic efficiency of ES Pair: higher specificity=E is more efficient at converting S to P
2. Max # this could be is 10^8 and 10^9 1/Ms

Question 11

Reversible inhibition: competitive inhibitors

Answer 11

1. 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

Question 12

Reversible inhibition: uncompetitive inhibitors

Answer 12

1. Bind to ES complex (don’t resemble substrate)
   1a. Lower Km, lower Vmax (new Vmax is apparent Vmax)
2. They bind to E after substrate is bound
3. Work better if substrate and inhibitor concentrations are increased

Question 13

Reversible inhibition: mixed inhibitors

Answer 13

1. Bind to either ES complex or free E (don’t resemble substrate)
   1a. Lower or unchanged Km, lower Vmax
2. 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

Question 14

Michaelis menten equation + reversible inhibitor

Answer 14

1. Vo=((Vmax)([S])/((aKm) + (a’[S]))
   1a. Apparent Km=aKm/a’
   1b. Apparent Vmax=Vmax/a’
2. a> or = 1 and a’ >or = 1
   2a. Competative inhibiton: a’=1
   2b. Uncompetitive inhibition: a= 1

Question 15

Lineweaver-Burk Plots/double reciprocal plot

Answer 15

1. The reciprocal of michaelis menten equations:
   1a. 1/Vo=Km/((Vmax[S]) + 1/Vmax)
2. 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)

Question 16

Irreversible inhibitors

Answer 16

1. Form one or more stable bonds (covalent or not) with an enzyme which interrupts the normal function of the enzyme
2. Tend to be highly toxic

Question 17

Types of feedbacks from enzymes

Answer 17

1. Negative feedback/feedback inhibition: occurs when the product downstream inhibits the upstream reaction
2. Positive feedback: series of enzymatic reactions that stimulate the enzymes upstream (Ex: oxytocin in birth)

Question 18

Regulation by enzymes: allosteric effectors/modulators

Answer 18

1. 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)
2. Show sigmoidal saturation curve from cooperativity

Question 19

Regulation by enzymes: proteolytic cleavage (irreversible covalent modification)

Answer 19

1. Proenzymes (ex: zygomens) are proteolytically cleaved to be active

Question 20

Regulation by enzymes: control proteins

Answer 20

1. Protein subunits that bind to certain enzymes to activate or inhibit them
2. Ex: GPCR

Question 21

Regulation by enzymes: reversible covalent modification

Answer 21

1. Regulatory mechanism that occurs in many ways
2. 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)
3. Example: deamidation:
   3a. Amide of asparagine or glutamine is cyclicized which loses a NH3

Question 22

Enzyme classification

Answer 22

1. Ligament: joins 2 molecules (ex: synthetase)
2. Isomerase: moves functional groups from one position to another within a molecule (ex: mutase)
3. Lyases: breaks substrates apart without water (ex: decarboxylase, aldolase, synthase)
4. Hydrolase: hydrolysis (exergonic) to break down macromolecules with water (ex: phosphatase)
5. Oxidoreductase: transfers electrons from reluctant to oxidant (ex: dehydrogenase (uses NAD+, NADP+, FAD) , oxidase)
6. Transferase: moves functional groups from one molecule to another (ex: kinase, phosphorylase)

Question 23

Cooperativity

Answer 23

1. If H=1 : no cooperative
2. If H>1: Cooperative

Question 24

Independent vs dependant variable in science

Answer 24

1. Independent: x axis, unchanged, cause etc (time)
2. Dependant: y axis