Initially, substate conc. is proportional to rate. Eventually, as we continue adding substrate, rate levels off. This results in a rate being independent of substrate concentration. When it is independent, it is called the Vmax

Effect of temperature on rate

Michaelis Menton Equation

• substrate concentration greater than enzyme concentration (means E will always exist as ES)
• ES complex concentration does not change with time (steady state)

Km = (k-1 + k2)/k1 = (k-1/k1) = 1/2 Vmax

k2 is considered rate limiting (so it is so small it could drop out)

X and Y axes of Lineweaver Burk plot

• x = 1/S
• y = 1/Vo

• x int.- 1/Km
• y int.- 1/Vmax
• slope = Km/Vmax

irreversible

reversible (competitive, non-competitive, uncompetitive)

Mechanism of action and effect on LW plot of irreversible inhibitor

• mechanism of action (E + I → EI)
  • bind covalently to inhibitor and blocks active site and does not leave (ex: nerve gas, heavy metal ions)
  • portion of enzyme removed
• Effect on Lineweaver Burk plot
  • decreased Vmax (change in y intercept)
  • no change in Km (same x intercept)

Example of irreversible inhibitor: mechanism of action of organofluorophosphates (more specifically, diisopropylfluorophosphate)

1. react with Ser OH at active site of acetyl cholinesterase (AChE)
2. inhibits AChE (leads to continual nerve stimulation)

Normally, AChE breaks down ACh into choline and acetate.

• organoflurophosphates
  • sarine
  • diisopropylfluorophosphate

1. react during transition state of enzymatic reaction
2. active once it binds to active site
   1. enzyme thinks its a substrate, so begins catalysis
   2. this brings inhibitor into transition state
3. at transition state, the inhibitor is VERY ACTIVE
4. it will form covalent linkages at the active site
5. this results in inactivation of enzyme (committed suicide)

• mechanism of action 
  1. binds to enzyme at active site due to is structure being an analog of substrate
  2. EI complex has ability to dissociate as well
     1. this means we can overcome by increasing concentration of substrate
• LB plot effects
  • Vmax SAME
  • Km increases

Examples of competitive inhibitors

• malonic acid inhibits succinate dehydrogenase
  • malonic acid only differs by one CH2 group with succinate
• sulfanilamides ("sulfa drugs") inhibit PABA
  • sulfa groups have sulfonic group in place of carboxylic acid

Example of competitive inhibitor- mechanism of action of malonic acid inhibiting SDH

• as a substrate analog of PABA, block synthesis of folic acid in bacteria (doesnt affect humans since we can't convert PABA to folic acid)
  • folic acid is vitamin

• mechanism of action
  1. involves a two substrate reaction
  2. measure disappearance of S1 as inhibitor completes for S2
  3. changin concentration of S1 has no effect on inhibition
• Effect on LB plot
  • Vmax decreased
  • Km same

• substrate availability
• enzyme concentration
• temperature
• pH

General substrate conc. for most enzymes in cell

• absolute amount
• relative amount (amount in active form)
  • proteolysis
  • allosteric modification
  • covalent modification

• proteolysis activates a large inactive precursor molecule
• ex: pepsinogen in the presence of HCl is ativated to pepsin + hexapeptide

Different forms of covalent modification to affect enzyme activity

• methylation
• phosphorylation
• adenylation
• uridylation
• ADP ribosylation

Example of covalent modification- General mechanism of action of phosphorylation to activate/inactive enzyme

1. enzyme receives phosphate via ATP hydroylsis via protein kinase
2. enzyme gives up phosphate via phosphoprotein phosphatase

Whether active with or without phosphate varies enzyme to enzyme

• mechanism of action (violates Michaelis Mento kinetics as shown by sigmoidal S x Vo curve)
  1. compound bind to enzyme at site other than the active site
  2. leads to confirmational change in enzyme
     1. can cause increase or decrease affinity for substrate
• structure- have subunits (usually regulatory or catalytic subunits)

• Positive will increase Vo (higher rate at given substrate concentration) while negative decreases Vo (lower rate at given substrate concentration)