Week 1 - Enzymology Flashcards
(30 cards)
kcat (Turnover rate)
- Fastest speed of an enzyme
- kcat determines the Vmax
- The rate varies substantially among enzymes.
- Reflects the catalytic power of the enzyme (relative to the uncatalyzed rate).
∆Go
the difference in energy between the substrate and the product is the standard state free energy of the reaction: ∆Go.
Recall that ∆G should be negative for a spontaneous forward direction, is positive for a spontaneous reverse reaction, and 0 at equilibrium.
Also remember, because catalysts are unchanged during the reaction, they cannot put energy into the reaction. So the basic energy difference between substrate and product, the ∆G, remains unchanged. The enzyme can change the rate of the reaction, but not how favorable it is.
pH sensitivy for enzymes
Enzymes evolved to work optimally at a relevant pH:
– Low pH for pepsin to act in the stomach
– Moderately low pH for lysosomal enzymes
– Alkaline phosphatase – very stable enzyme can survive environmental extremes
- Stomach enzymes have a low pH optimum because of the low pH environment. Lyzosomal enzymes likewise have a pH optimum below neutrality.
- Alkaline phosphatase, a general phosphatase, works best at high pH.
- Most cytosolic enzymes will have pH optima near neutrality.
How enzymes speed up reaction?
Catalysts speed up the reaction.
So, effectively, catalysts lower the barrier (smaller barrier, faster rate).
One of the means is that they actually stabilize the transition state itself,
Mechanisms of Catalysis
- Specific substrate interactions
- binding transition state optimally
- providing covalent chemistry
- providing acid base chemistry
Specific substrate interactions
that is, only the substrate binds effectively. Other compounds do not bind as well, even if similar in structure.
- Substrate specificity is critical for catalysts so as not to generate mis-reactions on the wrong compounds.
- It is critical to provide a locally higher concentration of the substrates.
- It is critical to put the substrate in apposition, or near, the critical reactive residues, either on the enzyme or on another substrate (or both).
Binding the transition state optimally
- Refer to the transition state diagram shown earlier where the enzyme reduces the barrier to the reaction. (Slide 7)
- This second catalytic mechanism is just the manifestation of this diagram.
- If the enzyme binds the transition state well, even better than substrate, what will happen is that substrate will bind, and then deform a little, because of the binding interactions. This deformation will bend the substrate into something that looks more like the transition state. This effectively lowers the barrier to achieve the transition state and is caused by the binding interactions with the enzyme.
- So, properly positioned residues can stabilize the transition state and lower the barrier to reaction.
Mechanisms of Catalysis Hexokinase
- Enzymes arehighly specific for their substrates
- Even very similar compounds are typically excluded from the reaction
- This is critical for function in Biology
- Specificity is achieved not only by specific binding but also through subsequent changes in protein structure.
Mechanism of Chymotrypsin
These enzymes show features of lock and key binding for specificity, Of induced fit binding for transition state stabilization
Of Covalent chemistry
And both acid and base catalyzed chemistry.
– Enzyme localized in
the small intestines
– Cleaves peptide after aromatic residues.
Oxido-reductases
- Example: Alcohol Dehydrogenase
- Detoxifies ethanol.
- Uses NAD+ as a cofactor or FAD
- An example of an oxidation
- Ultimate product will be acetate which is metabolized.
- Facilitate redox chemistry.
- Often involve NAD or FAD
NAD requiring enzymes are called dehydrogenases (if you see this name, you know its an NAD requiring oxido-reductase).
- The example at the right is lactate dehydrogenase, an enzyme that converts pyruvate to lactate during anaerobic metabolism in muscle, and reverses the reaction in the liver
Transferases
Transferases move a group from one molecule to another.
A common example is kinases: They transfer a phosphate group from one molecule (ATP typically), to another. Example is protein kinase A, which will catalyze the transfer of phosphate from ATP, to regulated proteins.
– Example:GlycerolKinase
– Primes Glycerol for conversion to glucose in the liver during gluconeogenesis
Hydrolases
These use water addition to break a chemical bond. Proteins in this class include those that break peptide and ester bonds. For example, proteases, esterases, lipases.
Typically one molecule is cleaved into two new products.
Also included are nucleases, phosphodiesterases, helicases, DNA glycosylases. There are many others.
Example: Protease, Esterases, Proteases, Sugar hydrolases, Ether hydrolases
Key:additionofwater.
NOT hydroxylases
Lyases
Catalyze cleavage of C-C bonds, C-O bonds, C-N bonds, C-S bonds, and C-halide bonds. Also P-O bonds.
Decarboxylases are one example. Dehydratases are another (remove water; breaks C-O bond).
General examples:
- Dehydratases
- Decarboxylases
Isomerases
As the name implies, isomerases catalyze isomerization reactions, often among stereoisomers to racemize them.
The example of phosphoglucomutase, shows isomerization of glucose-6-phosphate to glucose- 1-phosphate. This is a readily reversible enzyme that functions near equilibrium.
Isomerases typically have one substrate and one product.
Ligases
Catalyze the formation of bonds: C-O, C-S, C-N, C-C and others.
The example shown is glutamine synthetase which adds ammonium to the amino acid glutamate.
This is an important reaction for the movement of nitrogen within the body.
– Example: Glutamine Synthetase
– Catalyzes transfer of ammonium to Glutamate
– Glutamine is a major nitrogen carrier in the blood
Coenzyme A
Cofactors help facilitate reactions where unique reactivity is required, or reactivity not readily supplied by the canonical set of 20 amino acids.
Used as an acyl carrier and is involved in many catabolic and anabolic reactions.
competitive inhibition on LB curve
Effects of a competitive inhibitor are readily seen as changes in a Lw-B plot Vmax is unchanged
Km is increased
Overall: this is a slope effect. You can see that in the equation – only the slope is affected, not the y-intercept.
Competition can be “reversed” by adding more substrate to out-compete the inhibitor – thus you get the same Vmax if you add enough substrate.
Because you have to add more substrate to get to the same activity, the apparent Km is increased (higher concentration).
competitive inhibition
- Competitive inhibitors bind the active site
- Competitive inhibitors do not react and are not substrates, typically.
- Competitive inhibitors typically have structural similarity to the actual substrate.
- Competitive inhibitors have key differences from the substrate that makes them unreactive
or less reactive to the enzyme.
• This example shows competition between two substrates, with distinctly different Km values.
statins
Statins are competitive inhibitors of HMG-CoA reductase.
Example: Pravastatin competes with HMG-CoA for the active site of HMG-CoA reductase.
Cholesterol is made in liver from acetate.
HMG-CoA reductase is the rate-limiting step in
cholesterol synthesis.
Statins competitively inhibit this enzyme and plasma cholesterol levels are reduced.
non competitive inhibition
Noncompetitive Inhibition (Can also be activation)
Occurs by binding of an inhibitor, I, outside the active site.
The binding of I does not interfere directly with substrate
The binding of I likely influences the conformation of the enzyme to reduce (or enhance) activity.
The principal change is in Vmax.
This is because adding I essentially just takes some of the enzyme out of action, effectively reducing the amount of active enzyme.
non competitive inhibition on LB plot
Vmax changes (is reduced for an inhibitor. Make sure you recall that in a double reciprocal plot, a higher value on the y-axis means a lower Vmax). Km is unaltered. The slope of the plot still changes.
