Active-Site Directed Irreversible Inhibitors and Proteolytic Enzymes

BCMB 301 ALAB 6B:

Trypsin and -ChymotrypsinProteolytic EnzymesSerine ProteasesSynthetic SubstratesInhibitors

Trypsin and -ChymotrypsinProteolytic EnzymesSerine ProteasesSynthetic SubstratesInhibitors

Trypsin and -ChymotrypsinProteolytic EnzymesSerine ProteasesSynthetic SubstratesInhibitorsBAPNA & GPPNA

Structurally resemble tripeptides

Enzymatic cleavage produces p-nitro anilide. Detectable at A405nm

Trypsin and -ChymotrypsinProteolytic EnzymesSerine ProteasesSynthetic SubstratesInhibitorsE + I E.I E-I

REAGENTS ARE POISONOUS.

WEAR GLOVES.

A. Substrate Specificity

Prepare 3 cuvettes

Buffer: assay bufferEnzyme: trypsin or chymotrypsinSubstrateBAPNAGPPNA100% DMF

SET REFMeasure A405nm every 15 seconds for 3 min

B. Inhibitor SpecificityPrepare 5 microfuge tubes:Incubate

Into a new microfuge tube:**from previous microfuge tubesSET REFMeasure A405nm every 15 sec for 3 min

WASH YOUR HANDS. THERE IS POISON ON THEM.

Results

Exchange data with your partner

Results Part A2 graphsOne for trypsin, one for chymotrypsin Part B2 graphsTable: % inhibition for each inhibitor% inhibition = |Vuninh Vinh| * 100 Vuninh

DiscussionNumber the questionsBe conciseAnswer the questions

Colored Eppendorf TubesBAPNA blueGPPNA pinkTrypsin purpleChymotrypsin - yellow

Chymotrypsin is an endoproteinase that cleaves on the carboxyl side of phenylalanine, tyrosine and tryptophan residues UNLESS they are followed by proline. Trypsin hydrolyzes peptide bons on the carboxyl side side of Lys and Arg residues.

*Serine proteases are characterized by a catalytic triad consisting of a Histidine, a Serine and an Aspartic Acid residue. These three residues end up close to one another in 3D space and during catalysis, electrons get juggled between them.Today we will be working with two synthetic substrates of trypsin and chymoptrypsin. BAPNA and GPPNA are synthesized molecules that satisfy two criteria: they fit into the active sites of trypsin or chymotrypsin, as the case may be, and upon reaction with the enzyme, both form a colored product that can be measured using a spectrophotometer.You will also be working with inhibitors of trypsin and chymotrypsin. Inhibitors, as you learned before, can also bind the enzyme. In the case of irreversible inhibitors a covalent bond forms between the enzyme and inhibitor, making it an irreversible step.*Before I get into the procedure I wanted to point that you will be working with reagent that are POISONOUS. That the inhibitors.Each of you will only be working with ONE of the enzymes but we want results for BOTH so make sure you swap raw data with your partners and then make your own graphs and tables.