Lecture 6 - Enzyme Mechanism & Regulation Flashcards
What are the 6 major classes of enzymes?
- Oxidoreductases
- Transferases
- Hydrolases
- Lyases
- Isomerases
- Ligases
What is a transferase?
Transfers functional groups from one substrate to another
like one that would do a nucleophilic substitution
What is a hydrolase?
Enzyme which catalyzes a hydrolysis reaction, cleaving the substrate with the addition of water (OH to one, H to the other)
What is a lyase?
Enzyme which catalyzes a lysis reaction without the use of water
What is an isomerase?
Enzymes catalyzing isomerization reactions by rearranging atoms in a molecule
What is a ligase?
Enzymes using ATP to ligate two molecules together. I.e. DNA ligase
What are the two models for the enzyme substrate complex?
- Lock and key model - substrate fits the active site perfectly
- Induced fit model - active site fits the transition state to the product perfectly
What are orientation and proximity effects?
Substrates are bound and held in optimum orientation for reaction to occur
How can substrate strain play a part in enzyme activity?
Enzyme imposes strain or distortion on the substrate molecule that makes it easy to cleave the bond
What is the passive vs active role of metal ion catalysis?
- Passive role: Attract / neutralize negatively substrate material. For instance, Mg+2 is required for ATP to band an enzyme active site.
- Active role: Increase the reactivity of groups involved in catalysis. For instance, Zn+2 coordinate of water and side chain atoms in the active site of carboxypeptidase A.
What is meant by covalent catalysis?
Forming and breaking covalent bonds to the enzyme in order to catalyze a reaction. For instance, in chymotrypsin, Enzymatic Serine-OH group is a better nucleophile than water, and greatly speeds up proteolysis.
What is a common example of protein denaturation?
Denaturation of albumen in egg whites, which causes cysteine to form disulfide bond crosslinks.
How does substrate concentration differ in vivo vs in vitro?
In vivo: [S] is about equal to Km
In vitro: [S] is about 30-50 fold greater than Km to assess Vmax
What is the Lineweaver-Burk plot and how is it derived?
Double-reciprocal plot, derived by taking the reciprocal of the Michaelis Menten formula.
Y-intercept is: 1/Vmax
Slope is Km/Vmax
X-intercept is: -1/Km
What are the three types of reversible enzyme inhibition?
- Competitive
- Noncompetitive
- Uncompetitive
How are the Km apparent and Vmax changed in competitive inhibition? How does this appear in Lineweaver Burk?
Km increases - Relatively more substrate is needed to reach half Vmax, since inhibitor is sometimes binding
Vmax stays the same - you can add enough substrate to make Enzyme-Inhibitor Complex formation unlikely
Y intercept (1/Vmax) does not change X intercept (-1/Km) moves more towards zero as [I] increases
What is non-competitive inhibition and how does the affect the apparent Vmax and Km?
Inhibitor binds allosteric site, does not interfere with substrate binding, but slows enzyme. This happens equally well with free enzyme or ES complex.
Vmax decreases - enzyme cannot work as fast due to presence of [ESI] complex
Km is unchanged - competitive and uncompetitive effects of a noncompetitive inhibitor are equal. Any increase in Km from the binding of the inhibitor to free enzyme will be compensated by a decrease in Km from the uncompetitive stabilization of the ESI complex
Why is it called noncompetitive?
Noncompetitive inhibitors do not bind at the active site, and are not competing for the same site. Thus, increasing [S] will not overcome inhibition, since they are not competing for binding. As long as inhibitor is present, true Vmax will never be achieved since [ES] will always be less than [E]subT (some will be ESI)
How do irreversible inhibitors work?
They covalently bond the free enzyme, taking that enzyme out of action
What kinetic profiles do irreversible inhibitors have and why?
Same as non-competitive inhibitors, because [ES] will always be less than [E]subT, because some enzymes will be [EI] due to irreversible binding to inhibitor. However, remaining enzymes have the same Km (same [S] needed to reach their lowered Vmax)
What is uncompetitive inhibition and how does it influence Vmax and Km?
The inhibitor binds to ES complex and stabilizes it. It’s “uncompetitive” because it actually works synergistically with substrate binding, and helps lower Km
Vmax is decreased since [ES] < [E]subT, and some enzyme is tied up as ESI.
Km is lowered by making is harder for ES to dissociate, since it stabilizes it. Initial binding is just as easy, but once bound, it’s less likely to go back to E+S, and just says ESI.
