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Biochemistry I Lecture 20 Oct 17, 20051Lecture 20: Analysis of Enzyme Inhibition & Protein PurificationReading in Campbell: Chapter 6.7, chapter 520.1 Summary of Analysis of InhibitionA competitive inhibitor binds to the substrate-binding pocket of E with a dissociation constant of† KI. By competing with substrate for binding to the same site in E, a competitive inhibitoreffectively increases the KM of the enzyme-catalyzed reaction. Because it does not bind to theES complex, a competitive inhibitor does not affect VMAX. a, the degree to which a competitiveinhibitor increases KM, is obtained from the ratio of the slopes (slope (+ inhibitor) / slope (–inhibitor)) on a double reciprocal plot. From a, † KI can be calculated if the inhibitor concentrationis known.† KI=[E ][I][EI]† 1v=aKmVMAX1[S]+1VMAX† a= 1 +[I]KIA noncompetitive inhibitor binds to both E and ES at a site distinct from the substrate binding site.Although a noncompetitive inhibitor usually binds to E and ES at the same site, it does so withdiffering affinities. It binds E with a dissociation constant of † KI and ES with a dissociationconstant of † KI'. Binding of a noncompetitive inhibitor to E and ES allosterically inhibits bothsubstrate binding and catalysis, respectively. Thus both KM and VMAX are affected. a, thedegree to which a noncompetitive inhibitor increases KM, is obtained from the ratio of the slopes(just like for a competitive inhibitor). a’, the degree to which a noncompetitive inhibitor decreasesVMAX, is obtained from the ratio of the y-ints (y-int (+ inhibitor) / y-int (- inhibitor)). † KI and † KI 'can be calculated from a and a’, respectively, if the inhibitor concentration is known.† KI=[E ][I][EI]† KI '=[ES][I][ESI]† 1v=aKmVMAX1[S]+a'VMAX† a= 1 +[I]KI† a'= 1 +[I]KI 'Biochemistry I Lecture 20 Oct 17, 20052Sample Analysis:20.2 Protein PurificationReading: Campbell Chapter 5Key Terms:• Specific Activity• Fold purification• Purification Scheme• Chromatographic StepsConcept of a Purification Scheme:A purification scheme usually begins with a crude mixture of cellular proteins, referred to as alysate. The lysate is treated in a series of physical steps or processes. Each step separates amixture of proteins into two or more fractions. Fractions that contain the protein or enzyme ofinterest are retained for the next step of the purification scheme while the other fraction(s) arediscarded until the protein is deemed to be pure. The entire sequential process is referred to as apurification scheme.The actual separation steps are based on different physical/chemical properties ofproteins:• Solubility in different salts (ammonium sulfate)• Size• Charge• Hydrophobicity• Binding to Specific LigandsBiochemistry I Lecture 20 Oct 17, 20053How to monitor purity: It is essential to have some method to evaluate fold-purification or fold-enrichment, otherwise how do you know whether a specific step in the purification scheme hasbeen successful in increasing the purity of the desired protein?Amount of target enzyme: It is essential to have some method of determining the amount ofthe desired enzyme at any given step of the purification scheme. Consequently, the most criticalstep in any purification scheme is to develop a suitable assay for the enzyme that is beingpurified.Measuring purity during purification: The purity of the enzyme during the purification scheme isgenerally monitored by measuring the specific activity. Specific activity is defined as:Specific Activity: The units of activity (eg, VMAX in the case of an enzyme catalyzed reaction) forthe desired enzyme divided by the total amount of all protein species in the sample. Typical unitswould be mmol/sec/(mg total protein), where the mmol/sec refers to the amount of productproduced (mmol)/unit time. As the purification scheme progresses, the specific activity shouldincrease, reaching a maximum for the pure enzyme.Example Purification Scheme:The following scheme is an example of purification of a large (i.e. > 50,000 Da) protein that issoluble in 3M ammonium sulfate and has a positive charge at pH=7.0.1. Initially, we begin with 1 gram of total protein which contains 100 units of activity, giving aninitial specific activity of 0.1 units/mg.2. The final yield is 1 mg of purified material with a specific activity of 70 units/mg. Since thetotal activity of the final material was 70 units, the yield of this purification scheme is 70% (70units/100 units).Biochemistry I Lecture 20 Oct 17, 20054Example purification scheme: Garden beets were placed into a Waring blender to produce aninitial lysate for the purification of rubisco, an important plant enzyme. The following data wereobtained for each step of the purification:SampleUnits of Activitymmol product/secTotal Protein(mg)Specific Activitymmolproduct/sec/mg1. Crude lysate50,000 mmol/sec5,000 mg102. After separation bysolubility in ammoniumsulfate.45,000 mmol/sec900 mg503. After separation by size.40,000 mmol/sec800 mg504. After separation by charge.30,000 mmol/sec10 mg3000i ) Calculate the net % yield:ii) Calculate the increase in purity:iii) Which step in the above purification schemewas a complete waste of time? How do youknow?iv) Is the protein pure after the last step?Evaluating Final Purity:After the protein is pure, its purity can be monitored by: a) SDS-page gel electrophoresis.b) Mass spectrometry.c) Amino terminal sequencing.Separation using Column Chromatography:In most cases chromatography is performed in long glass tubes filled with a matrix or resin(particle size similar to a fine sand) that is completely immersed in a buffered salt solution. Themixture of proteins is added to the top of this column and buffer is allowed to flow through thecolumn. As the buffer flows through the column the mixture of proteins is drawn down throughthe column and interacts with the matrix or resin. The actual mode of separation depends on thenature of the resin. Usually several different chromatographic steps are performed with differentresins during a purification scheme. The actual order of separation methods will depend on theprotein being purified.Separation by Binding:• Anion exchange• Cation exchange• Hydrophobicity• Affinity chromatographyBiochemistry I Lecture 20 Oct 17, 20055TypeType of ResinPrincipal of SeparationHow to Elute the


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