2. The mature form of TEM-1 ß-lactamase, an enzyme of 290 amino acid residues that hydrolyzes penicillin antibiotics. The protein has the following polar amino acid side chains: 18 Arg; 11 Lys; 6 His; 4 Tyr; 16 Asp; 20 Glu; 8 Asn; 5 Gln. Approximate pKa values 1.5 Arginine 12.5 Lysine 10.0 Histidine 6.5 OD 280nm Tyrosine 10.0 Aspartate or Glutamate 4.0 Terminal NH3* 8.0 Terminal COOH 4.0 0.0 A 0.01 0.10 KCI B Volume (mL) The chromatogram above shows a typical result in the purification of TEM-1 ß-lactamase overex- pressed in E. coli cells. The protein is eluted from an anion exchange column at pH 6.25 with an increasing gradient of KCI. Fraction A is shown to correspond to native enzyme. Since Fraction B has the same amino acid sequence for the first 10 residues, the conclusion is that this fraction represents enzyme in which side chains of glutamine or asparagine residues have been hydrolyzed (deamidated). (a)( ) What is the isoelectric point of the native enzyme? (b) ( ) Compared to the native protein, in which direction will the isoelectric point of the deamidated protein change? How will the change in isoelectric point affect the overall charge on the protein at pH 6.25?
2. The mature form of TEM-1 ß-lactamase, an enzyme of 290 amino acid residues that hydrolyzes penicillin antibiotics. The protein has the following polar amino acid side chains: 18 Arg; 11 Lys; 6 His; 4 Tyr; 16 Asp; 20 Glu; 8 Asn; 5 Gln. Approximate pKa values 1.5 Arginine 12.5 Lysine 10.0 Histidine 6.5 OD 280nm Tyrosine 10.0 Aspartate or Glutamate 4.0 Terminal NH3* 8.0 Terminal COOH 4.0 0.0 A 0.01 0.10 KCI B Volume (mL) The chromatogram above shows a typical result in the purification of TEM-1 ß-lactamase overex- pressed in E. coli cells. The protein is eluted from an anion exchange column at pH 6.25 with an increasing gradient of KCI. Fraction A is shown to correspond to native enzyme. Since Fraction B has the same amino acid sequence for the first 10 residues, the conclusion is that this fraction represents enzyme in which side chains of glutamine or asparagine residues have been hydrolyzed (deamidated). (a)( ) What is the isoelectric point of the native enzyme? (b) ( ) Compared to the native protein, in which direction will the isoelectric point of the deamidated protein change? How will the change in isoelectric point affect the overall charge on the protein at pH 6.25?
Biology: The Dynamic Science (MindTap Course List)
4th Edition
ISBN:9781305389892
Author:Peter J. Russell, Paul E. Hertz, Beverly McMillan
Publisher:Peter J. Russell, Paul E. Hertz, Beverly McMillan
Chapter4: Cells
Section: Chapter Questions
Problem 1ITD
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