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Chymotrypsin
- digestive enz in pancreas and released to SI
- serube protease
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What is diisopropylphophofluoridate (DIPF)?
group specific reagent that modifies Ser residues
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What is tosyl-L-phenylalanine chloromethyl ketone (TPCK)?
affinity label that is similar in structure to chymotrypsin substrates; covalently modifies His 57 when reacted with chymotrypsin
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What is N-acetyl-L-phenylalanin p-nitrophenyl ester?
chromogenic color substrate that reacts with chymotrypsin to form p-nitrophenolate, which has a yellow color
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What are the two phases of chymotrypsin catalysis?
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What is the burst phase?
in chymotrypsin catalysis
formation of acyl-enz int; concurrent with colored product formation (from what substrate?)
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What is the limiting step in chymotrypsin catalysis?
deacylation step in burst phase (takes long time for enz to be "reset")
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What is the catalytic triad?
- three residues involved in chymotrypsin catalysis:
- Ser 195
- His 57
- Asp 102
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Asp 102
- in catalytic triad of chymotrypsin catalysis
- orients His 57 by H bonding to the imidazole ring of His 57
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His 57
- in catalytic triad of chymotrypsin catalysis
- H bonds to Ser 195
- serves as general acid catalyst during hydrolization of peptide bond
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Asp 102
- in catalytic triad of chymotrypsin catalysis
- orients His 57
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What is meant by substrate specificity in relation to chymotrypsin catalysis?
chymotrypsin only cleaves bonds on residues w/ lg, phobic side chains...due to S1 binding pocket.
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What is the S1 binding pocket?
- in chymotrypsin
- only fits side chains like Phe, Tyr, and Trp; allows binding of adj peptide into AS of chymotrypsin
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What is meant by P1, P2, P3, Pn?
substrate side chain residues on N terminal side of bond to be cleaved
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What is meant by P1', P2', P3', Pn'?
substrate side chain residues on carboxyl side of bond to be cleaved
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What is meant by S1, S2, S3, Sn?
binding sites on protease corresponding to P1, P2, P3, Pn
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What is meant by S1', S2', S3', Sn'?
binding sites on protease corresponding to P1', P2', P3', Pn'
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What is necessary for cleavage in the active site of chymotrypsin?
appropriate bonding of S1 pocket to Phe, Tyr, or Trp
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What do trypsin, elastase, and chymotrypsin have in common?
- serine proteases that have:
- 40% seq idenity
- identical mechanism
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What is the S1 pocket specificity for chymotrypsin?
Phe, Tyr, Trp
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What is the S1 pocket specificity for trypsin?
- Lys, Arg
- *Arg present at bottom of pocket
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What is the S1 specificity of elastase?
- Ala, Ser
- *two Val residues allow only small side chains
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Subtilisin
- bacterial protease
- same catalytic mech as chymotrypsin but diff structure...convergent evol esempio
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Carboxypeptidase II
- bacterial protease
- same catalytic mech as chymotrypsin, but diff structure....esempio di convergent evolution
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What is common among ALL proteases?
stabilization of tetrahedral TS int
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What are examples of cysteine proteases?
- Papain (da papaia)
- caspases
- *both play roles in apoptosis (think immuno)
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How is the mech of cysteine proteases similar to serine proteases?
utilizes Cys and His residues in AS just as Ser proteases use Ser and His in AS
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What nucleophile is created during Cys protease mech?
S nucleophile: da His residue activating the Cys -SH
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What are examples of Aspartyl proteases?
- Renin: enz in BP reg
- Pepsin: dig enz
- HIV protease: req for HIV prog
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What does two-fold symmetry mean?
in aspartyl proteases; contain two fold symmetry that is result of two copies of a gene evolving into a single chain enz...results in pair of aspartate residues in AS
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What are examples of metalloproteases?
- thermolysin: bact enz
- carboxypeptidase A: dig enz
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What is unique about metalloproteases?
contain a metal ion (usually zinc)
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What is the ligand for the metalloprotease metal ion?
water molecule
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What are some of the characteristics of HIV proteases?
- asp protease
- dimer of identical subunites, each with an Asp residue
- has flaps which close in on binding pcoket after the substrate has bound
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What is crixivan?
HIV protease
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How does crixivan function?
- analog of tetrahedral TS int
- specificity to HIV protease e' da chem groups on drug that interact w/ S2, S1', S2' binding sites on HIV enz
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How does the HIV protease-crixivan complex work?
- adopts conf similar to two fold symmetry of protease; AS covered by flaps
- *two carbonyl groups on inhibitor make water mediated H bonds to an NH group in each flap (unique to HIV proteases); OH group of central alcohol is analog of negatively charged oxygen in tetrahedral TS and interacts w/ two Asp side chains at AS
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