biochem hmwk 3

An amino acid has pKas of 1.96 (α-carboxyl), 8.65 (α-aminium), and 10.70 (δ-aminium). What is the net charge when pH = pK1?

+1.5

• At pH = pK1: 50% of α-carboxyl groups are protonated (neutral), 50% are deprotonated (-1)

• Essentially all α-aminium and δ-aminium groups are protonated (+1 each)

• Net charge = 0.5(+2) + 0.5(+1) = +1.5

What is the one-letter code for the amino acid with a methyl group as its side chain?

A (Alanine)

• Alanine has -CH₃ as its side chain

• "Obvious" classification - first letter of amino acid name

An anion-exchange resin has functional groups that are positively charged. A scientist wants to purify a protein with an isoelectric point (pI) of 8.1. A sample containing the target protein has been equilibrated in a buffer at pH 7.0. When the biochemist adds the sample to the anion-exchange column, will the protein of interest bind to the resin?

No, it will not bind

• At pH < pI, protein has net positive charge

• Anion-exchange resin is positively charged

• Like charges repel, so protein won't bind

What is the pI of leucine given pK1 = 2.36 (α-carboxyl) and pK2 = 9.60 (α-aminium)?

5.98

• For amino acids with no ionizable side chains: pI = (pK1 + pK2)/2

• pI = (2.36 + 9.60)/2 = 5.98

What is the isoelectric point (pI) of the following peptide: NH2-Glu-Cys-His-Lys-Arg-COOH? Use these pKa values: N-terminal aminium = 8, C-terminal carboxyl = 3.5, Asp = 4.0, Glu = 4.0, His = 6, Cys = 8.4, Tyr = 10, Lys = 10, Arg = 12.5.

Slightly below pH 7 At pH 7: 90.9% of N-terminal aminium groups carry positive charge, essentially all glutamate residues are negatively charged, 3.83% of cysteine residues are negatively charged, 9.09% of histidine residues are positively charged, essentially all lysine residues are positively charged, and C-terminal carboxyl groups are negatively charged for an overall net charge of −0.038. The pI is about 6.92.

Consider the peptide shown: NH2-Phe-Tyr-Ser-Lys-Val-COOH. What is the amino acid sequence using one-letter code?

FYSKV

What would be the approximate net charge of the peptide NH2-Asp-Glu-COOH at pH = 8? Use these pKa values: N-terminal aminium = 8, C-terminal carboxyl = 3.5, Asp = 4.0, Glu = 4.0.

-2.5 At pH = 8, half of the N-terminal aminium groups would be protonated (positive) and half would be neutral. The side chains of aspartate and glutamate would be unprotonated (negative), and the C-terminal carboxyl would be unprotonated (negative) for a −2.5 charge overall.

At physiological pH, how many carbons, hydrogens, and nitrogens are predominantly present in the side chain of tryptophan?

9 carbons, 8 hydrogens, 1 nitrogen

Samples of histidine, leucine, and glycine are equilibrated in pH = 6.0 buffer and applied to an electrophoresis strip. When voltage is applied, how will the samples migrate?

Histidine will move toward the cathode, and leucine and glycine will more or less stay in the middle. Histidine will have a net positive charge at pH 6 (approximately +0.5) and thus will move toward the cathode; leucine and glycine will both be close to their pI's at pH 6, and thus both will more or less stay in the middle.

Linus Pauling determined that the peptide bond had approximately ____% double-bond character.

~40%

The two pKas of a simple amino acid are 2.01 and 9.43 for the α-carboxyl and α-aminium groups, respectively. At pH 9.83, what percentage of amino acid molecules are in the zwitterion form?

28% At pH 9.83, essentially all the α-carboxyl groups are in the carboxylate form (COO−), so the percent that is zwitterionic will correspond to the percent of the α-amino groups that are in the aminium form (NH3+), which is 100 times 10^(pKa – pH)/(1 + 10^(pKa – pH)).

A single-subunit protein has folded such that the side chains of an aspartate residue and a cysteine residue end up close to each other (~3 – 4 Å apart). What is the likely effect on the pKa of the cysteine side chain compared to the free amino acid?

The pKa of the cysteine side chain would likely be perturbed to a higher value. A higher pKa for this side chain would prevent the dissociation of a proton from the neutral thiol form to yield the negatively charged thiolate form at lower pH values, which would prevent two like charges from occupying a similar space.

If the α-amino group of histidine was protonated, we would refer to it as an α-__________ group.

aminium When an amine (amino) functional group is protonated it is called an aminium group.

If the peptide KPLRW was incubated with the protease chymotrypsin, how many fragments would result?

No cleavage would occur Chymotrypsin catalyzes hydrolysis of peptide bonds on the C-terminal side of amino acid residues that have aromatic side chains, but the only aromatic residue (tryptophan) is at the C-terminal end of the peptide, and thus cleavage would not occur.

A pentapeptide contains tryptophan, lysine, arginine, serine, and threonine. When incubated with trypsin, a dipeptide and tripeptide result. The tripeptide contains tryptophan, arginine, and serine. When incubated with chymotrypsin, a tripeptide and dipeptide result. The dipeptide contains arginine and serine. What is the sequence?

TKWRS Because trypsin digestion resulted in two fragments, either lysine or arginine must be the last amino acid. Because the dipeptide from chymotrypsin digestion contained arginine and serine, the third amino acid must be tryptophan, so the last three residues are tryptophan, serine, and arginine. The first two amino acids are threonine and lysine.

Is the Fischer projection structure shown D- or L-cysteine? (α-carbon at intersection, horizontal bonds toward viewer, vertical bonds away from viewer)

D-cysteine Convert the structure to compare with D-glyceraldehyde reference. If it takes an even number of interchanges to convert the compound to have the same relative configuration as D-glyceraldehyde, then it's D-configuration. If odd number of interchanges, then it's L-configuration.

For a generalized amino acid Fischer projection (R group not H, lower priority than carboxylate), what is the relative configuration (D or L)?

L The amino acid has the L-configuration in the Relative system of nomenclature.

For a generalized amino acid Fischer projection (R group not H, lower priority than carboxylate), what is the absolute configuration (R or S)?

S The amino acid has the S configuration in the absolute system of nomenclature.

Is the structure shown L-isoleucine with absolute configuration 2S, 3S?

Yes, the structure has the absolute configuration 2S, 3S, so it is L-isoleucine.

To which of the 20 standard amino acids does a titration curve with three buffering regions at pKa ~2, ~9.5, and ~12.5 correspond?

R (Arginine) The titration curve shows three buffering regions. The first has pKa ~2, the second has pKa ~9.5, and the third has pKa ~12.5. The only amino acid whose pKas match these three values is arginine.

To which of the 20 standard amino acids does a titration curve with three buffering regions at pKa ~2, ~9.5, and ~12.5 correspond?

R (Arginine) The titration curve shows three buffering regions. The first has pKa ~2, the second has pKa ~9.5, and the third has pKa ~12.5. The only amino acid whose pKas match these three values is arginine.

For a generalized amino acid Fischer projection (R group not H, lower priority than carboxylate), what is the relative configuration (D or L)?

D Convert the structure to compare with D-glyceraldehyde reference. If it takes an even number of interchanges (exchanges of two groups) to match D-glyceraldehyde configuration, then it's D-configuration. If odd number of interchanges, then it's L-configuration.

: For a generalized amino acid Fischer projection (R group not H, lower priority than carboxylate), what is the absolute configuration (R or S)?

R Mentally view the molecule with the lowest priority group (H) behind the α-carbon. Label remaining groups 1, 2, 3 in priority order. If tracing 1→2→3 goes clockwise, it has R absolute configuration; if counterclockwise, it has S absolute configuration.

To which of the 20 standard amino acids does a titration curve with three buffering regions at pKa ~2, ~6, and ~9.5 correspond?

H (Histidine) The titration curve shows three buffering regions: first at pKa ~2, second at pKa ~6, and third at pKa ~9.5. The only amino acid whose pKas match these three values is histidine.

An amino acid has an alpha-carboxyl, an alpha-aminium group, and a delta-carboxyl group. The respective pKas of these three groups are 2.14, 9.77, and 4.21. What is the isoelectric point (pI) of this amino acid?

3.18 The pI for this amino acid is the average of pK1 and pKR because these two pKas "bracket" the neutral species. pI = (2.14 + 4.21)/2 = 3.18

What is the one-letter code for the amino acid that has as its side chain two methylenes (CH₂s) followed by a primary amide group?

Q The side chain of glutamine (Gln, Q) contains two methylenes (CH₂s) followed by a primary amide group. Memory aid: think Qtamine for Glutamine.

A cation-exchange resin has functional groups that are negatively charged. A scientist wants to purify a protein with an isoelectric point (pI) of 6.1. A sample containing the target protein has been equilibrated in a buffer at pH 7.0. When the biochemist adds the sample to the cation-exchange column, will the protein of interest bind to the resin?

No, it will not bind to the resin. When the pH is higher than the pI, the protein will have a net negative charge, and because the resin (a cation-exchange resin) has a negative charge, the like charges of the protein and resin will repel and thus the protein will not bind to the resin.

What is the isoelectric point (pI) of the following peptide: NH₂-Asn-Val-Lys-COOH? Use these pKa values: N-terminal aminium = 8, C-terminal carboxyl = 3.5, Asp = 4.0, Glu = 4.0, His = 6, Cys = 8.4, Tyr = 10, Lys = 10, Arg = 12.5.

9.0 The isoelectric point is the average of the pKas of the side chain of Lys and the N-terminal aminium group. The net charge at pH 7 would be close to +1; thus electrical neutrality occurs at the average of these two pKas: (10 + 8)/2 = 9.0

Consider the peptide shown: NH₂-Tyr-Phe-Val-Lys-Cys-COOH. What is the amino acid sequence using one-letter code?

YFVKC