Chapter 5

Amino acid analysis of an oligopeptide 7 residues long gave \(\begin{array}{lllll}\text { Asp } & \text { Leu } & \text { Lys } & \text { Met } & \text { Phe } & \text { Tyr }\end{array}\) The following facts were observed: a. Trypsin treatment had no apparent effect. b. The phenylthiohydantoin released by Edman degradation was c. Brief chymotrypsin treatment yielded several products, including a dipeptide and a tetrapeptide. The amino acid composition of the tetrapeptide was Leu, Lys, and Met. d. Cyanogen bromide treatment yielded a dipeptide, a tetrapeptide, and free Lys. What is the amino acid sequence of this heptapeptide?

Amino acid analysis of a decapeptide revealed the presence of the following products: \(\begin{array}{lllll}\mathrm{NH}_{4}^{+} & \text {Asp } & \text { Glu } & \text { Tyr } & \text { Arg } \\ \text { Met } & \text { Pro } & \text { Lys } & \text { Ser } & \text { Phe }\end{array}\) The following facts were observed: a. Neither carboxypeptidase \(A\) or \(B\) treatment of the decapeptide had any effect. b. Trypsin treatment yielded two tetrapeptides and free Lys. c. Clostripain treatment yielded a tetrapeptide and a hexapeptide. d. Cyanogen bromide treatment yielded an octapeptide and a dipeptide of sequence NP (using the one-letter codes). e. Chymotrypsin treatment yielded two tripeptides and a tetrapeptide. The N-terminal chymotryptic peptide had a net charge of -1 at neutral \(\mathrm{pH}\) and a net charge of -3 at pH 12 f. One cycle of Edman degradation gave the PTH derivative What is the amino acid sequence of this decapeptide?

Amino acid analysis of an octapeptide revealed the following composition: \(\begin{array}{llllll}\text { 2 Arg } & \text { 1 Gly } & \text { 1 Met } & \text { 1 Trp } & \text { 1 Tyr } & \text { 1 Phe } & \text { 1 Lys }\end{array}\) The following facts were observed: a. Edman degradation gave b. CNBr treatment yielded a pentapeptide and a tripeptide containing phenylalanine. c. Chymotrypsin treatment yielded a tetrapeptide containing a C-terminal indole amino acid and two dipeptides. d. Trypsin treatment yielded a tetrapeptide, a dipeptide, and free Lys and Phe. e. Clostripain yielded a pentapeptide, a dipeptide, and free Phe. What is the amino acid sequence of this octapeptide?

Amino acid analysis of an oligopeptide containing nine residues revealed the presence of the following amino acids: \(\begin{array}{llllllll}\text { Arg } & \text { Cys } & \text { Gly } & \text { Leu } & \text { Met } & \text { Pro } & \text { Tyr } & \text { Val }\end{array}\) The following was found: a. Carboxypeptidase A treatment yielded no free amino acid. b. Edman analysis of the intact oligopeptide released c. Neither trypsin nor chymotrypsin treatment of the nonapeptide released smaller fragments. However, combined trypsin and chymotrypsin treatment liberated free Arg. d. CNBr treatment of the 8 -residue fragment left after combined trypsin and chymotrypsin action yielded a 6-residue fragment containing Cys, Gly, Pro, Tyr, and Val; and a dipeptide. e. Treatment of the 6 -residue fragment with \(\beta\) -mercaptoethanol yielded two tripeptides. Brief Edman analysis of the tripeptide mixture yielded only PTH-Cys. (The sequence of each tripeptide, as read from the N-terminal end, is alphabetical if the one-letter designation for amino acids is used.) What is the amino acid sequence of this nonapeptide?

Describe the synthesis of the dipeptide Lys-Ala by Merrifield's solidphase chemical method of peptide synthesis. What pitfalls might be encountered if you attempted to add a leucine residue to Lys-Ala to make a tripeptide?

Phosphoproteins are formed when a phosphate group is esterified to an - OH group of a Ser, Thr, or Tyr side chain. At typical cellular \(\mathrm{pH}\) values, this phosphate group bears two negative charges \(-\mathrm{OPO}_{3}^{2-} .\) Compare this side-chain modification to the 20 side chains of the common amino acids found in proteins and comment on the novel properties that it introduces into side-chain possibilities.

A quantitative study of the interaction of a protein with its ligand yielded the following results: Ligand concentration \(1 \quad 2 \quad 3 \quad 4 \quad 5 \quad 6 \quad 9 \quad 12\) \((m M)\) \(\nu\) (moles of ligand \(\begin{array}{lllllll}0.28 & 0.45 & 0.56 & 0.60 & 0.71 & 0.75 & 0.79 & 0.83\end{array}\) bound per mole of protein Plot a graph of [L] versus \(\nu .\) Determine \(K_{\mathrm{D}},\) the dissociation constant for the interaction between the protein and its ligand, from the graph.

The human insulin receptor substrate- 1 (IRS-1) is designated protein \(\mathrm{P} 35568\) in the protein knowledge base on the ExPASy Web site (http://us.expasy.org/). Go to the PeptideMass tool on this Web site and use it to see the results of trypsin digestion of IRS-1. How many amino acids does IRS-1 have? What is the average molecular mass of IRS-1? What is the amino acid sequence of the tryptic peptide of IRS-1 that has a mass of \(1741.9629 ?\)

Proteases such as trypsin and chymotrypsin cleave proteins at different sites, but both use the same reaction mechanism. Based on your knowledge of organic chemistry, suggest a "universal" protease reaction mechanism for hydrolysis of the peptide bond.