Enzymes Flashcards
Describe basic characteristics of enzymes:
- catalyst
- active site
Catalyst – accelerates chemical rxn; participates in rxn but not consumed; returns to original state after chemical rxn; can be as anything from a small compound to RNA to a protein enzyme; work by lowering the activation energy for a reaction to occur – does not change difference in free energy
Active Site – site of interaction; positions R-groups of a.a. or has prosthetic groups for appropriate interaction
- Describe Michaelis-Mentin kinetics
- Interpret a Michaelis-Menten curve for an enzyme reaction showing the relation between the substrate concentration and reaction rate
- Michaelis-Menten Equation
- At very high [S] near Vmax; enzyme working at “turnover number” (max rate enzyme can operate); Vmax constant for given enzyme
- Graph = Vmax vs. [S]
- Km = Michaelis-Menten constant = amount of substrate at which enzyme rxn is 1/2 Vmax
- Represents substrate conc
- Does not depend on enzyme con
- High affinity = lower Km
Differentiate between
- competitive
- non-competitive
- uncompetitive
- mixed enzyme inhibition
In competitive inhibition, Km increases because it takes more substrate (S) to reach 1/2 Vmax.
Inhibition can be completely overcome by increasing S, therefore Vmax is unchanged
Differentiate between
- competitive
- non-competitive
- uncompetitive
- mixed enzyme inhibition
Noncompetitive inhibitors bind outside the active site. This has the following effects:
- Binds to free enzyme (E) or enzyme-substrate (ES) complex
- Does not compete with substrate (S)
In these reactions, the enzyme cannot overcome inhibition by increasing substrate concentration. Km is unchanged Vmax decreases.
Differentiate between
- competitive
- non-competitive
- uncompetitive
- mixed enzyme inhibition
bind outside the active site. This has the following effects:
Binds to free enzyme (E) or enzyme-substrate (ES) complex
Does not compete with substrate (S)
In these reactions, the enzyme cannot overcome inhibition by increasing substrate concentration. Km is unchanged Vmax decreases.
Differentiate between
- competitive
- non-competitive
- uncompetitive
- mixed enzyme inhibition
- Like non-competitive inhibitor but Ki,1 does not equal Ki,2
- Both slope and y-intersect ae altered by the inhibitor; lines cross at a point that’s not on the axis
Explain what is shown in a Lineweaver-Burk plot and describe how it can distinguish between competitive, non-competitive, uncompetitive, and mixed enzyme inhibition (graph examples shown in other cards) – EQUATION on this card
Lineweaver Burke Plot equation – given as a form of y=mx+b
(1/V) = [(Km/Vmax) * (1/[S]) ] + (1/Vmax)
The slope of the Lineweaver-Burk plot is Km/Vmax
The y-intercept is 1/Vmax
The x-intercept is -1/Km
Describe how serine proteases function
Serine Protease Mechanism
- Substrate protein binds to serine protease
- Shift in active site
- Acyl enzyme intermediate formed
- Tetrahedral intermediate
- 1st peptide product released
- Another tetrahedral intermediate
- Release of 2nd peptide chain
- Serine protease back in original state
protease: hydrolyzes peptide bond in proteins
Define the role of coagulation in the process of hemostasis
- Prothrombin found in blood as inactive zymogen
- Factor Xa (serine protease) cleaves prothrombin to become active thrombin
- Thrombin = serine protease; cleaves arg-gly peptide bonds in protein substrates; cleaves fibrinogen into fibrin
- Fibrinogen has 6 polypeptide chains (α2β2γ2); Thrombin cleaves fibrinogen: 1 bond in each α chain and 1 bond in each β chain releasing fibrinopeptides A and B.
- New terminal ends on alpha and beta of fibrin can polymerize to form a soft clot
- Soft clots to hard clots; Factor XIIIa catalyzes cross linking between fibrin molecules in soft clots to form polymerized fibrin (hard clots)
Describe the two coagulation pathways
Intrinsic Clotting Pathway
- Cascade of serine protease cleavage events; rapid, localized production of thrombin
- Polyphosphate = signal that blood vessel wall has ruptured
- Helper proteins = Factor Va and Factor VIIIa
Extrinsic Clotting Pathway
- Cascade of serine protease cleavage events; rapid, localized production of thrombin
- Adventitia tissue factor initiates pathway; signal of damage (VII to VIIa)
Explain the importance of factor X, prothrombin, and fibrinogen to the coagulation process (also antithrombin III)
Factor X: serine protease; can be activated in either intrinsic or extrinsic pathway to cleave prothrombin into thrombin
Prothrombin: the zymogen form of thrombin; found in blood and undergoes proteolytic cleavage by Factor X
Fibrinogen: zymogen of fibrin; cleavaged by the serine protease thrombin
antithrombin III: serpin that inhibits thrombin (primarily) and Factors Xa, IXa, XIa, and XIIa
- serpin: Serine Protease Inhibitors; bind to and inhibit serine proteases
