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98 Cards in this Set
- Front
- Back
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How common are enzymes in the human body?
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Nearly all chemical rxns in the human body are sped up by enzymes.
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Enzymes _____________ the thermodynamics of a rxn....
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Enzymes DO NOT AFFECT the thermodynamics of a reaction.
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Do enzymes affect the equilibrium concentrations of reactants and products?
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Enzymes DO NOT affect the equilibrium concentrations of reactants and products.
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What affect do enzymes have on the direction of a reaction?
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Enzymes do not determine the net direction of a reaction.
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What do enzymes do to the rate of a rxn?
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Enzymes speed up the rate at which a specific reaction proceeds toward equilibrium.
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In what direction do enzymes speed up the reaction?
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Enzymes speed up the reaction equally in both directions.
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How do enzymes facilitate reactions?
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Enzymes reduce the energy needed (energy of activation) to force a reaction through the transition state (ts).
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What are the six classes of enzymes?
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1. oxidoreductases
2. transferases 3. isomerases 4. hydrolases 5. lyases 6. ligases |
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What does the Michaelis-Menten reaction describe?
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Michaelis-Menten equation describes the rate of a given reaction based on the concentration of the substrate (S).
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Tell me about Km...
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Km is the [S] at which enzyme activity is 1/2 max. Thus, v=1/2Vmax when [S] = Km
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When will the enzyme be maxed out?
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When substrate is plentiful ([S]>>Km), the enzyme will be maxed out. v=Vmax
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If two enzymes catalyze the same reaction but have different values for Km, which one will work better at low [S]?
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The enzyme with the lower Km will work better at low [S].
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For hexokinase (Km 0.15mM) and glucokinase (Km 10mM), which one works better at low [glucose]?
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Hexokinase works better at lower [glucose]. Lower Km
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What is kcat?
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kcat is the "turnover number" or maximal activity for a single enzyme.
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What is the fastest known enzyme?
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Catalase (2 H2O2-->2 H2O + O2) where kcat = 40 x 10^6/sec
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Lineweaver-Burk also known as...
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double reciprocal plot
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Lineweaver-Burk reciprocal plot; x and y axis...
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1/v vs 1/[S]
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What does the y axis on a L-B plot represent?
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1/Vmax
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What does the x axis on a L-B plot represent?
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-1/Km
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What does irreversible inhibition often involve?
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Irreversible inhibition often involves a suicide inhibitor which becomes covalently bound to the enzyme and prevents further activity.
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What is an example of a suicide inhibitor in irreversible inhibition?
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Penicillin-irreversibly inactivates glycopeptide transpeptidase, an enzyme that cross-links bacterial peptidoglycan cell walls.
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What is the enzyme inactivated by Penicillin?
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glycopeptide transpeptidase
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Aspirin/acetylsalicylate:
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irreversible inhibitor of cyclooxygenase (cox-1 & 2)
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What do competitive inhibitors do?
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Competitive inhibitors compete with S (&P) for the active site. Competitive inhibitors increase Km, but do not alter Vmax because saturating levels of S swamp out the inhibitor.
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How do competitive inhibitors affect Km?
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Competitive inhibitors increase Km.
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How do competitive inhibitors affect Vmax?
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Competitive inhibitors have no effect on Vmax because saturating levels of S swamp out the inhibitor.
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What are the statins?
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The statins are competitive inhibitors of HMG CoA Reductase, a key enzyme in the pathway that synthesizes cholesterol.
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Lipitor and Crestor are cholesterol lowering drugs that...
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are competitive inhibitors of HMG CoA Reductase, an enzyme in the pathway that synthesizes cholesterol.
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How do noncompetitive inhibitors work?
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Noncompetitive inhibitors can bind the enzyme at the same time as S but reduce the turnover rate.
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What effect do noncompetitive inhibitors have on Km?
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Noncompetitive inhibitors do not effect Km
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What effect do noncompetitive inhibitors on Vmax?
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Vmax is reduced
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What do mixed mode and uncompetitive inhibitors do to Km and Vmax?
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They alter both Km and Vmax
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Are all enzymes active all the time?
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In biological pathways, some of the enzymes are unregulated and are active essentially all of the time; a few key enzymes will be regulated.
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For unregulated enzymes, what can you say about the reactions they catalyze?
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Unregulated enzymes are active essentially all of the time and the reactions they catalyze will almost always be close to equilibrium.
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Are most enzymes regulated?
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A few key enzymes will be regulated and the reaction will be prevented from reaching equilibrium.
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What are the four primary modes of enzyme regulation?
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1. reversible allosteric inhibitors
2. covalent modifications (phosphorylation) 3. induction or repression of gene expression 4. protein degradation |
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What are zymogens? How are they activated?
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Zymogens are inactive protein precursors that are activated by proteolysis.
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What can plasma enzyme activities indicate?
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Possible organ or tissue damage.
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What is elevated alanine aminotransferase (ALT) activity consistent with?
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elevated ALT is consistent with liver damage
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What is elevated creatine kinase (CK)associated with?
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Elevated CK is associated with muscle damage.
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How can you distinguish between skeletal muscle CK and cardiac CK?
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You can analyze the isozymes present in the plasma.
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What is the only tissue with appreciable levels of the mixed isozyme (BM)?
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myocardium
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How can the three isozymes of CK be separated?
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Use native protein electrophoresis because the three different protein dimers (BB, BM, MM) all have a different net charge so all three move through the electric field at different rates.
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What are the three isozymes of CK?
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BB, BM, MM
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What would SDS-PAGE electrophoresis do to the dimeric complexes of CK?
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SDS-PAGE electrophoresis would separate the dimeric complexes into single monomers because the SDS unfolds the protein thile B-ME reduces any disulfide bonds.
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What is the utility of the Western blot?
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Proteins separated on any kind of electrophoresis can be transferred to membranes and probed with antibody (Ab) to detect a specific protein.
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What is isoelectric focusing (IEF)?
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IEF occurs when a protein is electrophoresed in a gel that contains a pH gradient; when the protein reaches the pH equal to its pI, there will be no net charge on the protein and it will stop moving.
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What do the most popular 2D protein gels use? Why?
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The most popular protein gels use:
IEF followed by SDS PAGE More than 1,000 proteins can be separated using this approach. |
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What does temperature do to enzyme activity?
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Temperature increases enzyme activity until the protein starts to unfold (denature).
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At what temp do many human enzymes begin to denature?
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40C
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What pH do enzymes work best at?
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Enzymes operate in a particular pH window. Protease pepsin works well at pH 1-2, but is nearly inactive at neutral pH of small intestine pH 6-7. protease Trypsin is nearly inactive in the stomach but maximally active at pH 6-7.
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Structural protein example for zymogen:
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procollagen-->collagen
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Peptide hormone example for zymogen:
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proinsulin-->insulin
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Protease example for zymogen:
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trypsinogen-->trypsin
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Where is pepsinogen synthesized?
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stomach
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Where is chymotrypsinogen synthesized?
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pancreas
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Where is trypsinogen synthesized?
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pancreas
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Where is procarboxypeptidase A and B synthesized?
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pancreas
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Where is proelastase synthesized?
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pancreas
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What is the specificity of pepsin?
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broad
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What is the specificity of chymotrypsin?
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Phe, Tyr, Trp
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What is the specificity of trypsin?
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Lys, Arg
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What is the specificity of carboxypeptidase A, B?
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Cleaves C-term aa, broad specificity
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What is the specificity of elastase?
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Gly, Ala, (Ser, Cys)
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How is pepsinogen activated?
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autocatalysis
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What is the pathway for activation of the pancreatic zymogens?
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Enteropeptidase activates trypsinogen. Trypsin activates all 4 pancreatic zymogens
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What three digestive enzymes are serine proteases?
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1. chymotrypsin
2. trypsin 3. elastase |
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Chymotrypsin, trypsin, and elastase are all what type of enzyme?
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serine proteases
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What is nearly the same in the 3 serine proteases (chymotrypsin, trypsin, elastase)?
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nearly superimposable
same catalytic triad (Asp102, His57, Ser195) same catalytic mechanism |
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How do the 3 serine proteases trypsin, chymotrypsin, and elastase differ?
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1. each cleaves after a different subset of amino acids
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What is "the secret" behind serine proteases?
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The ability of the catalytic triad to abstract a proton from serine and make it a powerful nucleophile.
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Serine protease sequence of events (basic):
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1. (poly)peptide substrate binds
2. Ser attacks carbonyl carbon 3. Cleavage of peptide bon 4. H2O binds and attacks |
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Where is preproinsulin synthesized?
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Insulin is synthesized at the RE of pancreatic beta cells
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Where is the signal sequence of preproinsulin removed?
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in the lumen
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Where is the C-peptide of proinsulin removed?
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Golgi apparatus
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What happens to proinsulin in the Golgi apparatus?
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The c-peptide is removed.
Mature insulin and the c-peptide are packaged into secretory granules. |
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What enhances granule secretion of mature insulin and c-peptide by the beta cells of the pancreas?
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increased plasma glucose
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What function does the c-peptide from insulin serve for the organism?
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Binds to receptors and upregulates TS
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What function does the c-protein from insulin serve to the medical practitioner?
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Tells how much insulin is being made.
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What is the reason behind inactive zymogens?
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allows most or all of the components of a cascade to be present but inactive until needed
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Pepsinogen is activated by...
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autocatalysis due to low pH in the stomach
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What stimulates the secretion of pancreatic zymogens?
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CCK-produced when peptides and lipids show up in the stomach
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Food in stomach->enzyme release and activation cascade...
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-low pH in the stomach activates pepsinogen
-peptides and lipids in the stomach stimulate CCK -CCK stimulates secretion of pancreatic zymogens enteropeptidase->^trypsin->^activation of pancreatic zymogens |
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Serine proteases... what does that mean?
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serine at the active site
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What percentage of the aa sequence is conserved between the 3 serine proteases?
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40%
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How do the three serine proteases differ?
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1. pocket next to the active site is different
2. each cleaves after a different subset of amino acids |
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Do aspartic acid proteases form acyl intermediates?
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NO!!! Aspartic acid proteases DON'T form acyl intermediates with substrate.
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How do aspartic acid proteases work?
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they use 2 asp to hyrolyze a peptide bond
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What are the 4 basic steps in aspartic acid protease action?
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1. Substrate binds and then H2O attacks.
2. Proton shifts on Asp. 3. Cleavage of peptide bond. 4. Products leave and proton shifts-start anew. |
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What do you know about the AIDS virus protease?
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HIV-1 protease (p11) is an Asp protease.
It has unusual specificity (Tyr-Pro) |
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What type of inhibitor is the HIV protease inhibitor?
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Transition state analog that mimics the intermediate state-binds stronger than the substrate->good competitive inhibitor.
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Who first proposed transition state analogs?
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Linus Pauling
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Why are transition state analogs so effective?
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TS analogs bind the enzyme tighter than substrates or products.
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What group of proteins is responsible for the blood clotting cascade? Where are they?
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serine proteases-plasma-as zymogens
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Catalytic triad?
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Asp, His, Ser
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Why are transition state analogs so effective?
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TS analogs bind the enzyme tighter than substrates or products.
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What group of proteins is responsible for the blood clotting cascade? Where are they?
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serine proteases-plasma-as zymogens
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Catalytic triad?
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Asp, His, Ser
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