Wk 2 TBL 4 Enzyme Kinetics Flashcards
What is the pyrimidine biosynthetic pathway?
Pyrimidine synthesis (cytosine, thymine, and uracil)
What does OMP decarboxylase (UMP synthase) do?
Removes a CO2 in the pyrimidine biosynthesis pathway from OMP to make UMP
What is unique about the OMP decarboxylase enzyme?
1/2 life of the reaction is > 78,000,000 years w/o the enzyme
W/ enzyme: 1/2 life is <0.02 s
What is special about the delta-ALA synthase?
- 1st step in heme synthesis, puts glycine and succinyl-CoA together to make ALA
- derivative of B6 needed at active site
- linked to X-linked sideroblastic anemia
What can often cure X-linked sideroblastic anemia?
High doses of vit B6
Why do high doses of vit B6 often work to cure X-linked sideroblastic anemia?
The cofactor B6 vitamin derivative that’s part of ALA synthase helps stabilize the structure to help it work well enough to keep the heme synthesis pathway operating
What happens at enzyme active sites?
Substrates bind to the enzyme active sites where chemical transformations occur to create the product
How do active site AAs work?
Often act as acids and bases in the reaction
-enzyme activity often very pH sensitive
What dicates the reaction specificity?
The active site structure
Why are cofactors or coenzymes often necessary?
Reactions req chem capabilities not available from AA so the cofactors and coenzymes provide them
What is enzyme catalysis?
lowering the free energy of the transition state
-enzymes alter the kinetics (rates) but NOT the thermodynamics (the equilibrium b/w substrates and products) of a reaction
What does k stand for?
kinetics/rate of reaction
kcat = rate with catalysis
What do interactions w/ enzymes do to the free energy of transition states?
Lower the free energy
Why could a fever potentially lead to hypoglycemia in a neonate?
The rate of chemical and enzyme-catalyzed reactions increases ~12% per increase in degrees C, so a fever -> increased metabolic rate
What is rate-limiting for a reaction?
Binding of a substrate
What is rate-limiting for a reaction?
Binding of a substrate
What shape is a saturation curve?
hyperbolic
Example of non-enzymatic reaction with different kinetics
HbA + glucose -> HbA1c
-linear reaction rate - as substrate increases, the rate of formation increases
Yente analogy to enzyme
What happens to the max rate if more enzymes are recruited?
If double the enzymes, double the max rate (Vmax)
What is the equation that fits a typical enzyme curve?
michaelis menten equation for hyperbolic curve:
What is Vmax?
maximal velocity - rate no longer increases when it is saturated w/ substrate
What is the Km?
substrate concentration that gives 1/2Vmax
=approx measure of the affinity for an enzyme for its substrate
-lower concentrations -> tighter binding
-independent of enzyme concentration
What values change w/ changed enzyme concentration?
Vmax but not Km
When is the rate of reaction most sensitive to changes in [substrate]?
Below Km
-the curve is steeper
The rate sensitivity to [substrate] changes has caused what to evolve regarding enzymes?
To have Km values > the physiologic [substrate]
What is the kcat?
Turnover rate for an enzyme
=product/time
-value is independent of [enzyme] present in a reaction or cell
-a per enzyme molecule rate (=#marriages each Yente can arrange per year)
What values does enzyme concentration affect?
Vmax but not kcat
What are 2 common mechanisms for enzyme regulation other than by gene expression?
- post-translational modification
- reversible binding of activators or inhibitors
What is a competitive inhibitor?
Compete w/ substrate for binding
-Vmax stays same (w/ high enough [substrate], enzyme can still achieve same max rate
-Km increases b/c takes more substrate to get to 1/2Vmax
What would the double-reciprocal plot of competitive inhibition look like?
What does the slope of double reciprocal plot look like?
slope = Km/Vmax
What is the intercept of a double-reciprocal plot?
intercept = 1/Vmax
What does the double reciprocal plot look like w/ competitive inhibitor?
lines Cross = Competitive inhibitor
What happens to enzyme kinetics when noncompetitive/uncompetitive inhibitors bind?
Vmax ALWAYS decreased
Km may or may not be affected
What is a chemically stable analog of a tansition state?
An inert molecule that does not react with the enzyme even though it has binding interactions like the transition state
=competitive inhibitors
Summary
What is the transition state?
the most unstable or high energy state along the reaction pathway
Do catalysts/enzymes alter the equilibrium of a reaction?
No b/c they are regenerated after each reaction cycle
What is the major source of free energy used to lower the activation energy barrier?
Binding energy, which is free energy released from interactions b/w substrates and enzymes in the complex with weak, noncovalent bonds
What are active sites most complemetary to?
The transition states of the reactions they catalyze, not the substrates directly
Besides activation energy, what else can enzymes affect?
They can reduce the entropy that substrates must overcome to form a product
-binding energy holds the substrates in the proper position
At what temp do enzymes typically begin to unfold?
45 degrees C
What is the reaction rate increase with enzymes for each 1 degree C increase?
~10%
How are steady-state kinetics performed?
Use a small (and constant) concentration of enzyme and measure how fast product is formed when differing concentrations of substrates are used.
-Generates a rate vs [substrate] curve
Why are steady-state kinetics performed?
To understand how enzymes fxn and what happens when someone inherits a genetic variant in a gene encoding an enzyme
How do rate vs [substrate] curves compare with and w/o enzymes?
curvilinear vs linear
