Ch. 7 and 8 Flashcards
What is the significance of P50 for Hb?
P50 is the concentration of O2 required to saturate 50% of the binding sites
What is the site of O2 binding for Mb and Hb?
the Heme group
What does a lower value of P50 indicate?
a binding site with a high O2 binding affinity
What does a higher value of P50 indicate?
a binding site with a low O2 binding affinity
What are the 4 subunits of Hb?
2 identical alpha-globins and 2 identical beta-globins
What are some characteristics of the T state of Hb?
Low O2 binding affinity
High P50
more stabilizing non-covalent interactions
What are some characteristics of the R state of Hb?
High O2 binding affinity
Low P50
less stabilizing non-covalent interactions compared to the T state
What is the idea of cooperativity of Hb?
each binding of an O2 molecule lowers the P50 for that specific Hb
Does the R state have more or fewer Fe2+-O2 (iron-oxygen) bonds than the T state?
More
Does the T state have more or fewer Fe2+-O2 (iron-oxygen) bonds than the R state?
Fewer
What are the 3 negative effectors of Hb that stabilize the T state?
CO2, H+, and 2,3-BPG
What is the positive effector that stabilizes the R state of Hb?
O2
Which state (R or T) is favored in the lungs?
R state favored, binding of O2 and release of CO2, H+, and BPG
Which state (R or T) is favored in the capillaries?
T state favored, binding of CO2, H+ and BPG, release of O2
Is the T state stabilized or destabilized when going from a higher to lower pH?
stabilized
Explain the Perutz mechanism
O2 binds, Fe2+ ion is pulled into the plane of the heme, this pulls the F-helix down, causes steric strain, which is relieved in the T-state
Explain the molecular basis for HbS (Sickle cell anemia)
A Glu –> Val mutation at Beta 6
Hb forms long rod-like structures that distort and can rupture the membrane of the RBCs
How do you recognize a general acid catalyst?
transfers a H+ to an atom
How do you recognize a general base catalyst?
removes a H+ from an atom
How do you recognize a covalent catalyst?
formation of a covalent E-S adduct that alters the rxn pathway
How do you recognize an electrostatic catalyst?
occurs when the enzyme active site stabilizes the transition state of the reaction by forming electrostatic interactions with the substrate
which is faster: intramolecular interactions or intermolecular interactions?
always intramolecular
define Km
the substrate concentration at which the Vo is 1/2 of Vmax
What is Km analogous to?
P50
define Kcat
measures the rate of the catalytic process (how rapidly S –> P)
what is the significance of kcat/Km?
a measure of enzyme efficiency and substrate specificity
What structural features of an enzyme are most likely to affect Km?
the binding of S (selectivity of “target”)
What structural features of an enzyme are most likely to affect kcat?
Stabilization of the transition state
What does the slope of the Lineweaver-Burk plot represent?
Km/Vmax
What does the y-intercept of the Lineweaver-Burk plot represent?
1/Vmax
How do you recognize competitive inhibition?
slope changes, no change in y-intercept
How do you recognize uncompetitive inhibition?
y-intercept changes, no change in slope
How do you recognize mixed inhibition?
changes in both y-intercept and slope
define a competitive inhibitor - where does it bind?
inhibitor competes with the substrate - binds and blocks the active site
how can you overcome competitive inhibition?
very high levels of substrate concentration
define an uncompetitive inhibitor - where does it bind?
binds at a site on the enzyme different from the substrate and diminishes the enzyme’s catalytic activity
define a mixed inhibitor - where does it bind?
binds to both the active site or a site other than the active site
How do Km and Vmax change with a competitive inhibitor?
Km increases, Vmax unchanged
How do Km and Vmax change with an uncompetitive inhibitor?
Km and Vmax both reduce
How do Km and Vmax change with a mixed inhibitor?
Vmax decreases
Km typically increases
What type of modification do irreversible inhibitors make?
a covalent modification
How do you increase flux? (movement of material through a pathway)
- increase substrate concentration
- remove products as they are generated
What are the features of the “committed step” in a pathway
usually early in the pathway, delta G «_space;0 which allows for regulation of “off” and “on”
What are the features of the R state of an enzyme?
higher binding affinity for substrate
lower Km
higher kcat
activators (+ effectors) stabilize
What are the features of the T state of an enzyme?
lower binding affinity for substrate
higher Km
lower kcat
inhibitors (- effectors) stabilize
What are the 2 things an enzyme must do to be an effective catalyst?
Bind to Substrate
Stabilize the transition state
What amino acids can be phosphorylated?
Thr, Ser, Tyr
What enzyme does phosphorylation?
Kinase
What enzyme removes a phosphate group?
Phosphatase
What type of modification is phosphorylation?
reversible covalent modification
explain an irreversible covalent modification
usually the result of proteolytic cleavage
enzymes produced in the inactive form, they mature and become fully active as a result of proteolysis
