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BioChem: Enzyme Kinetics


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What two things do enzymes do?
1) speed up slow reactions
2) catalyze otherwise impossible reactions
What binds a small molecule to a Protein?
-Ionic bonds
-Hydrophobic interaction
-Van der Waals forces
(very sensitive to interatomic distance)
What are 6 classes of enzymes?
1) oxidoreductases
2) transferases
3) hydrolases
4) lyases
5) Isomerases
6) Ligases
What reaction type is: oxidoreductases?
ex; Lactate Dehydrogenase
What reaction type is: Transferases?
-Group Transfer
ex; nucleoside monophosphate kinase (NMP Kinase)
What reaction type is: Hydrolases?
ex; Chymotrypsin
What reaction type is: Lyases?
Addition or removal of groups to form double bonds
ex; fumarase
What reaction type is: Isomerases?
-Intramolecular group transfer
ex; Triose phosphate isomerase
What reaction type is: Ligases
-Ligation of two substrates at the expense of ATP
ex; Aminoacyl-tRNA synthetase
What does alcholol dehydrogenase do?
-Catalyze the reduction of Acetaldehyde to ethanol
-Systematic name is EC
What does NMP kinase do?
-It transfers a phosphoryl group from ATP to NMP to form NDP, hence it is a transferase-group 2
How do you calculate velocity for a linear plot of kinetic data?
What is this called?
Vo= (Vmax[S])/(Km + [S]
OR 1/Vo= (Km/Vmax)x(1/[S])+(1/Vmax)
*Called the Michaelis-Menton Equation
What is the name of the plot one would use for a Uni-Uni enzyme?
-double reciprocal plot OR
-Lineweaver-Burk Plot
What are 5 reasons for finding Km?
-Km comparisons
-different substrates
-Ligand induced changes in Km
-intracellular [S]
-Enzyme assay
What is Aspariginase used for?
What variation is there?
-Leukemia treatment
-aspariginases with high or low Km's were ineffective
Where is hexokinase found?
-In the muscles of man and animals
-also in the liver (along with glucokinase
What is the main difference between hexokinase and glucokinase in terms of what they use?
-hexokinase (low Km of 37uM)will work with many hexoses, whereas glucokinase only uses glucose (high Km of 10mM)
How does hexokinase work in muscle?
-works as fast as it can, near Vmax
-any glucose or hexose taken up by muscle is phosphorylated and locked into the muscle
How does hexokinase work in the liver?
-hexokinase can not respond to blood glucose increase, but it can work with glucose in the liver for catabolic needs (of liver)
-after a meal, liver imports more glucose to store as glycogen
Where is lactate dehydrogenase found?
-in muscles (and therefor the heart)
How does muscle LDH work?
-muscle LDH has a high Km for pyruvate. As pyruvate increases, more lactate is formed and fatigue results
How does heart LDH work?
-since heart shouldn't get fatigued, LDH has a low Km for pyruvate in the heart:
-can't respond to increased pyruvate
-high pyruvate completely inhibits LDH in heart
What are enzyme inhibitors used for?
-establish nature of reactants
-establish nature of binding sites
-stablish the specificity of the enzyme
-determine the reaction mechanism
What is the basis for rational pharmacology and chemotherapy?
-Drugs work as selective inhibitors by natural (or synthetic) compounds
-Inhibition of enzymes lies at the root of the mode of action of many drugs
What are the two classes of inhibitor classes?
What happens to Vmax and Km in an irreversible inhibitor reacton?
Vmax goes down and Km is unchanged
What affect do heavy metals have on proteins?
-since this is in the inhibitor section, it inhibits it!
-to be precise, metals inactivate proteins
What is a benefit of irreversible inhibitors?
-can identify imprtant groups in the enzyme
What is an example of how an irreversible inhibitor is used?
-Iodoacetate treatment of RNAase at pH 5.5 inactivates an enzyme
-Look for alkylation on His 12 or 119
-If there, these histidines are important in the active site of the enzyme
What is Acetylcholine Esterase?
-an enzymatic catalyst that is the site of action of organophosphate nerve gasses
How does a competitive inhibitor work?
-When inhibitor is added, reaction is pulled to the left, increasing Km (dissociation constant)
-If you add enough S to push all the enzymes to ES, you can get product still and Vmax is unchanged
What are the different types of reversible enzyme inhibitors?
-nonclassical competitive
Where do the lines intersect for each type of reversible inhibitor
-Competative: Y-axis
-uncompetitive: parallel
-noncompetative: X-axis
-mixed/nonclassical: intersect anywhere but the x or y axis
How does a Noncompetitive inhibitor work?
-no effect on binding of S so Km is unchanged
-can not force all of enzyme to ES, so Vmax is decreased
How does an Uncompetitive Inhibitor work?
-Pulls equation to the right, so Km decreases
-Can not drive all enzyme to ES, so Vmax decreases
How does a mixed type inhibitor work?
-effects binding site, so Km increased
-If I is present, you will always have ESI, hence Vmax decreased

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