From Biolk483


Exam 1 Summary

  • no questions on cell biology stuff
  • know all structures, even those barely mentioned
  • go get exam from him if you'd like it

Proteins continued

Enzyme kinetics


  • There are two types of inhibitors: irreversible, and reversible (which is made up of competitive and non-competitive)
Irreversible Inhibitors
  • These inhibitors bind at the active site and cannot be taken off
  • Example:
    • di-isopropyl flourophosphate on chymotrypsin
    • It binds to serine 195 and stops the enzyme from catalyzing.
    • Can be used to identify the active site amino acids.
Reversible Inhibitors
  • The more inhibitor is added the less the enzyme works.
Competitive Inhibitors
  • Usually, these inhibitors look like the substrate that the enzyme is supposed to bind with.
  • Example: Succinate dehydrogenase
    • Succinate + FAD -> Fumarate (which is oxidized) + FADH2 (which is reduced)
    • If we put malonate in as an inhibitor, it binds as would succinate but electrons cannot be used for redox reaction because malonate does not have the carbons in the middle of the chain to donate them.
  • So when you add competitive inhibitors:
    • Vmax does not change
    • Km increases
    • The slope of the reaction graph increases (because m = Km / Vmax)
  • It makes sense that Vmax doesn't change because we are not changing the enzyme's ability, only how often the right substrate lands in the active site.
Non-competitive Inhibitors
  • Does not bind where substrate binds.
  • Example:
    • Add a heavy metal --it usually binds to SH group
  • Note that these are not the same as allosteric affects
  • So when you add non-competitive inhibitors:
    • Vmax decreases
    • Km stays constant
    • The slope of the reaction graph increases (because m = Km / Vmax)
  • It makes sense that Vmax decreases because we are affecting the enzymes ability in a physical way.
  • Likewise, it makes sense that Km remains constant because we are not changing how often the proper substrate lands in the active site.
Multiple-substrate Enzymes and Inhibition
  • A + B -> P + Q
  • We can study them by making them pseudo first order by saturating them with one of the reagents (A or B)
  • The sequence of adding substrates can matter. If it does, it is called ordered, if it doesn't it is called random.
    • Random example: Creatin + ATP -> ADP + Creatin-p and it doesn't matter what order you add them.
    • Ordered example: Malate + NAD+ -> oxaloacetate + NADH+ and the NAD+ must be added first, then the malate.
Double Displacement Reactions
  • These are also called ping-pong reactions.
  • You bring in one substrate and release one product then repeat the process for each substrate.
  • Example: Aspartate transaminase
    • Aspartic acid + alpha-ketogluterate -> oxloacetate + glutamic acid
  1. Bind aspartic acid, then shift the amine to the pyridoxyl phosphate via shiff base formation.
  2. Oxloacetate leaves
  3. Alpha-ketogluterate binds, then transfer the amine from pyridoxyl phosphate to alpha-ketogluterate to produce glutamic acid.
  4. Glutamic acid leaves.

Enzyme Terminology


  • A method to follow reaction.
  • Look @ disappearance of substrate or formation of product.
  • Example: Chimotrypsin
    • Takes p-nitrophenylacetate (which is colorless) as substrate
    • When hydrolyzed, yellow is seen, so we can measure the amount of yellow appearing with spectrophotometer.

Enzyme Unit

  • Tells how much enzyme you have.
  • A unit of activity.
  • 1 micromol of substrate changed to product / minute.
  • Another similar unit is called the katal.
    • 1 mol of substrate changed to product / sec.

Specific Activity

  • Number of enzyme units / milligram of protein.
  • So, that is kind of like a proportion of your total sample that is actually your enzyme as opposed to all the other protein crap you isolated from the cell.
  • Example of use:
    • We grind up a cell
    • We see lots of activity (we are measuring the activity of the enzyme we want to know the specific activity of), but we're seeing lots of activity because there is a lot of the enzyme.
    • The task is to figure out how much activity each single enzymatic unit has --we want to purify down to just our enzyme while still seeing activity levels.
    • We make purification steps. Each step we take, we reduce the level of other crap around, but we also reduce the activity level of our target enzyme.
    • So we express specific activity in activity / milligram of protein material; therefore, as we get rid of the junk protein material, our specific activity measurement increases. We want to get the specific activity level to level out before we run out of enzyme material.
    • When the specific activity has leveled out, we can say that it is the true level.

Turnover Number

  • This is the number of substrates transformed / minute.
  • For example, one of the most efficient enzymes (that with the highest turnover number) has a turnover number of 36 million substrates to product per minute.
  • Now remember that some proteins are control points for other proteins, so if the control enzyme can make this many products per minute and the products can activate or deactivate other enzymes we can activate many different pathways extremely quickly.

Kinetic Measuring Problems

  1. Ribozymes can catalyze very select (and a very few) reactions. So, how do you measure a specific activity for this enzymatic process considering there is not protein material?
  2. Concentration of substrate from Mechanilis-Mentin model is too high; generally the number used is about 5-500 times larger than the concentration of substrate found _in vivo_ which is usually about 5 - 500 micromolar.
  3. Moonlighting: some proteins have more than one activity. For example, cytochrome C is part of the electron transport chain in the mitochondria but also activates apoptosis when outside the mitochondria.
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