7.014 Section Problem:There is a class of related enzymes called serine proteases that all use the same mechanism tocleave peptide bonds.Each member of this family cleaves protein substrates at a different location – that is, eachenzyme cleaves protein substrates after a different amino acid(s).Formally, they catalyze the following reaction:A-B-C-D-X-E-F-G-H + H2O --------------->A-B-C-D-X + E-F-G-H protein substrate protein fragment protein fragmentWhere A through G are any amino acid and X is one of the specific amino acids uniquelyrecognized by the enzyme. If this process repeats over and over, the substrate will be completelydegraded into small peptide fragments.Each member of the family cleaves after a specific amino acid of the substrate because that aminoacid is recognized by a binding pocket of the enzyme that is specifically designed to bind thatparticular amino acid.a) One of these protease enzymes, trypsin, cleaves after lys or arg. What would be the product(s)if each of the following molecules were treated with trypsin?i) leu-thr-phe-ala-ser ii) trp-tyr-lys-ala-phe iii) lys-arg-lys-argThe structures of three hypothetical proteases are shown below (not to scale – the actual enzymeis much larger than the binding pocket). If the amino acid side chain of the substrate binds well tothe “recognition pocket” the substrate will bind and be cleaved.ACTIVE SITE -peptide bond cleavageRECOGNITION POCKET -specific substrate regognitionGENERIC ENZYME:glyaspvalileglygly glyprotease Aprotease Bprotease Cglypheb) The specificities of each protease (the amino acids that it likes to cut after) are listed below.Match the enzyme with the specificity & explain.i) lysine, arginineii) phenylalanine, tryptophan, tyrosineiii) glycine, alaninec) How could you design a similar enzyme to cleave after aspartic acid?d) Speculate on the effect of changing the aspartic acid in protease B to a glutamic acid.e) There are three amino acids required for the active site to function and three amino acidsinvolved in substrate recognition – why then do these enzymes typically contain more than 200amino acids?f) Suppose you make a solution of protease A. A small sample taken when the solution was made(time = 0) is capable of cleaving 100 mmol of protein substrate per minute. This rate of substratecleavage is called the “activity” of the enzyme. You then take identical small samples from thissolution at regular intervals over the next few hours as the solution stands at room temperatureand measure their activity (the rate at which they cleave the substrate protein). You find that theactivity of the enzyme drops rapidly as time passes.However, if you add a large excess of casein (a protein found in milk which has noenzymatic activity), the protease loses its activity much more slowly. These data are sketchedbelow:activitytimeprotease A aloneprotease A + casein10000Explain these observations.g) If you monitor the reaction of any of these proteases as they degrade a protein, you observethat the protease does not cut all the recognition sites in the substrate at once – the recognitionsites on the surface of the substrate protein are the first to be cut. Explain.h) How might your answer to parts (f) and (g) be combined to design a long-lasting protease Amolecule?CNHHHHHHPeptide bondsCONHCCOHR1NHCCONCHHHR2R3Protein SynthesisSide chain, uniqueto each differntamino acidIndividual amino acidsare linked through thesegroups to form the backbone of the protein.GENERIC AMINO ACID:STRUCTURES OF AMINO ACIDSCCOOHNH3R-+VALINE(val)TYROSINE(tyr)TRYPTOPHAN(trp)THREONINE(thr)SERINE(ser)PROLINE(pro)PHENYLALANINE(phe)METHIONINE(met)LYSINE(lys)LEUCINE(leu)ISOLEUCINE(ile)HISTIDINE(his)GLYCINE(gly)GLUTAMINE(glN)GLUTAMIC ACID(glu)CCOOHNH3CH2OHHHHH-+CCOOHNH3CH2-+CCOOHNH3CHOHCH3-+CCOOHNH3CH2OH-++COO-CNCH2CH2CH2HHHCCOOHNH3CH2HHHHH-+CCOOHNH3CH2CH2SCH3-+CCOOHNH3CH2CH2CH2CH2NH3+-+CCOOHNH3CH2CHCH3CH3-+CCOOHNH3CHCH3CH2CH3-++CCOOHNH3CH2CNCNHHHH-+CCOOHNH3H-+CCOOHNH3CH2CH2OCNH2-+-CCOOHNH3CH2CH2OOC-+CYSTEINE(cys)CCOOHNH3CH2SH-+ASPARTIC ACID(asp)-CCOOHNH3CH2COO-+CCOOHNH3CH2CONH2-++CCOOHNH3CH2CH2CH2NHCNH2NH2-+CCOOHNH3CHCH3CH3-+CCOOHNH3CH3-+ASPARAGINE(asN)ARGININE(arg)ALANINE(ala)Solutions to: Enzymes/Protein Structurea) i) leu-thr-phe-ala-ser (unchanged) ii) trp-tyr-lys + ala-phe iii) 2 lys + 2 argACTIVE SITE -peptide bond cleavageRECOGNITION POCKET -specific substrate regognitionGENERIC ENZYME:glyaspvalileprotease Aprotease Cprotease Bglyphegly gly-peptide backbonelysine, argininephenylalanine, tryptophan, tyrosineglycine, alanineSchematics of substrates:Schematics of enzymes: (side chains shown in black)+long & + chargedlarge & hydrophobicsmallglyb) Matching them up:protease A - large open pocket. Could be lys/arg or phe/trp/tyr.protease B - large open pocket with (-) charge at bottom. Therefore, lys/arg, which means that protease A must cut after phe/trp/tyrprotease C - small pocket. Cuts after Gly, ala.c) Change the asp in the bottom of the pocket in protease B to a lys or arg.d) It might still bind lys or arg, but if the space in the pocket were constrained, there might not beenough room because glu is longer than asp.e) The others are required to hold the essential ones in place.f) Protease A is a protein, therefore other protease A molecules can cleave it and therebyinactivate it. Having casein around decreases the chance that a protease A molecule will cleave aprotease A molecule, because it will be more likely to cleave the more numerous caseinmolecules.g) The protease enzyme must be able to bind to the target amino acids. If they are buried insidethe target protein, the protease can't "see" them and therefore can't cut at them. Eventually, thestructure of the target protein gets so broken down that the inside amino acids are exposed to theprotease.h) To protect protease A from degradation by other protease A molecules, bury all thephe/trp/tyr inside the protein so that they cannot be recognized and
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