Peptides Introduction - Biochemistry PDF

PeptidesIntroductionChapter 1 discussed individual amino acids. Amino acids serve as the building blocks for an important class of molecules called polypeptides, which form when the amino group of one amino acid bonds with the carboxyl group of another amino acid. Two amino acids linked together in...

PeptidesIntroductionChapter 1 discussed individual amino acids. Amino acids serve as the building blocks for an important class of molecules called polypeptides, which form when the amino group of one amino acid bonds with the carboxyl group of another amino acid. Two amino acids linked together in this manner form a dipeptide. A third amino acid can then bond with the dipeptide to yield a tripeptide, which can link with another amino acid to form a tetrapeptide, and so on. This progression is shown in Figure 2.1.Figure 2.1 Peptides consist of two or more amino acids linked together.Peptides play several crucial roles in biological systems. This lesson explores the formation, degradation, and characteristics of peptides.2.1.01 Peptide Bond Formation and DegradationThe amino acids in a peptide are linked to each other through. A peptide bond forms when the carboxyl group of one amino acid backbone reacts with the amino group of another. In this process, the reacting amino acids undergo a net loss of one oxygen atom and two hydrogen atoms, which combine to form water. Therefore, peptide bond formation (Figure 2.2) is an example of a.Figure 2.2 Peptide bond formation is a condensation reaction.Because amino acids lose atoms during bond formation, they are not complete amino acids after the reaction. For this reason, the individual units in a polypeptide are called ---they are the residuals of amino acids. Chapter 2: Peptides and Proteins38Just as peptide bonds form with the accompanying release of a water molecule, a peptide bond can be broken when a water molecule is added across the bond (ie, water is consumed). This reaction, which is the reverse of condensation, is known as.Peptides, and therefore peptide bonds, are essential for life. However, under physiological conditions, peptide bond formation is thermodynamically unfavorable and peptide bond hydrolysis is favorable. In other words, without energy input, amino acids tend to remain unlinked. Consequently, energy input is required for peptide bond formation to occur in living cells.Once formed, peptide bonds remain intact for a long time, which allows them to perform their biological functions. This is because, although it is thermodynamically favorable for peptides to hydrolyze, peptide bond hydrolysis has a high. Therefore, hydrolysis is slow and peptide bonds are kinetically stable. For peptide bonds to be hydrolyzed at a significant rate, enzymes such as proteases or the are required.Concept Check 2.1The process of peptide bond formation is endothermic overall, yet bond formation itself is always an exothermic process. How can this apparent discrepancy be explained?2.1.02 Peptide Bond CharacteristicsPeptide bonds are. Therefore, the carbon atom is a carbonyl, is , and can participate in. The nitrogen atom contains a lone pair of electrons that also participates in resonance with the adjacent carbonyl.Consequently, the nitrogen atom is also sp2 hybridized, and the in the C=O double bond and the lone electrons on the nitrogen atom are all delocalized across the N, C, and O atoms of the amide group. This electron delocalization allows electrons to be present in a pi bond between the C and N atoms of the peptide bond, as shown in Figure 2.3.Figure 2.3 Resonance in peptides causes the peptide bond to include pi electrons, giving it partial double bond character.In other words, although the bond between C and N in a peptide is often drawn as a single bond, in reality, it exhibits significant double bond character. This double bond character has important effects on protein folding. Most importantly, atoms involved in a double bond always exhibit planar geometry ( in the case of the C and N atoms in a peptide bond). Together, these factors force all atoms linked to the carbon and nitrogen of the peptide bond into the same plane, as shown in Figure 2.4. Chapter 2: Peptides and Proteins39Figure 2.4 The C and N atoms in a peptide bond, and all atoms to which they are directly connected, are coplanar.In addition to this planarity, the partial double bond character of the peptide bond restricts rotation because double bonds unless the pi bond is temporarily broken. Therefore, the double bond character of peptide bonds limits the conformations that a peptide or protein can adopt. Most peptide bonds adopt the lower-energy trans (relative), or Z (absolute), configuration, which minimizes steric overlap of the groups on either side of the partial double bond (See Figure 2.5).However, because the side chain of connects back to its backbone amine group, both the cis (E) and trans (Z) configurations experience similar amounts of steric clashing. Consequently, the peptide bond preceding a proline residue can commonly be found either in the trans (Z) configuration or the cis (E) configuration. Chapter 2: Peptides and Proteins40Figure 2.5 The substituents around a peptide bond can be in either a cis or trans (E or Z) configuration. Most peptide bonds are trans (Z), while bonds preceding a proline can be either cis or trans.Although peptide bonds cannot rotate, the other bonds in a peptide backbone can. These are the bonds from the nitrogen to the α-carbon and from the α-carbon to the carbonyl.2.1.03 Peptide OrganizationThe backbone of each free amino acid contains one amino group and one carboxyl group. When two amino acids form a peptide bond, the carboxyl group of one amino acid and the amino group of the other are consumed and converted into a single. The resulting dipeptide has polarity (ie, the two ends of the dipeptide are distinct). One end contains a free amino group and is known as the amino terminus (N-terminus). The other end contains a free carboxyl group and is called the carboxy terminus (C-terminus).In biological systems, when another amino acid is added to the peptide, the amino group of the free amino acid reacts with the C-terminus of the growing peptide, consuming one amino group and one Chapter 2: Peptides and Proteins41carboxyl group to form a new peptide bond. The resulting tripeptide (or tetrapeptide, pentapeptide, etc.) still has one N-terminus and one C-terminus. The N- and C-termini of peptides of different lengths are shown in Figure 2.6.Figure 2.6 A peptide can react with an amino acid to become a longer peptide with more peptide bonds.Because biological systems synthesize the N-terminus of a peptide first and grow the C-terminus through the addition of amino acids afterward, amino acid sequences are, by convention, written and read from the N-terminus to the C-terminus.For example, the tripeptide ARE contains at the N-terminus, in the middle, and at the C-terminus. In contrast, the tripeptide ERA contains glutamate at the N-terminus, arginine in the middle, and alanine at the C-terminus. Therefore, although these tripeptides contain the same three amino acids, they are not identical and exhibit distinct chemical and biological properties. The structures of these peptides are shown in Figure 2.7.Figure 2.7 The peptides ARE and ERA contain the same three amino acid residues but have distinct structures. Chapter 2: Peptides and Proteins42Because the arrangement of amino acid residues in a peptide may change the peptide\'s properties, even relatively small peptides can exhibit substantial diversity. For example, a tripeptide that contains one A, one R, and one E can be arranged in six different ways (see Figure 2.8), which can be calculated as the of the number of residues being arranged (eg, 3! = 3-factorial = 3 × 2 × 1 = 6).Figure 2.8 The three amino acids in a tripeptide can be arranged in six different ways. Mathematically this is represented as 3! (3-factorial) or 3 × 2 × 1.This diversity is increased significantly by allowing a wider variety of amino acids. If all 20 proteinogenic amino acids are available to be used in a tripeptide (instead of only A, R, and E) and each can be used multiple times, the tripeptide may be arranged in different ways. A (ie, four residues) in which any of the 20 amino acids may be used can be arranged in 204 (160,000) different ways.Therefore, as more amino acids are used, the number of possible arrangements increases exponentially. This helps to explain how peptides and proteins can carry out diverse functions in living cells---the large number of possible arrangements gives rise to many possible sets of biochemical properties.The amino acid residues in the middle of a polypeptide (ie, those that are not at either end) no longer have backbone amino or carboxyl groups, nor do they have N- or C-termini. However, the peptide bond of an amino acid residue that is closer to the N-terminus is called the N-terminal side of the residue, and the other peptide bond is called the C-terminal side of the residue. This distinction is important in peptide bond hydrolysis because some protease enzymes cleave peptides on the N-terminal side of a specific residue, and some cleave on the C-terminal side. Chapter 2: Peptides and Proteins43Concept Check 2.2Consider the peptide MERGADLFN. The enzyme Arg-C proteinase cleaves peptide bonds on the C-terminal side of arginine, whereas the enzyme Asp-N endopeptidase cleaves peptide bonds on the N-terminal side of aspartate. Two samples of MERGADLFN are prepared. Sample 1 is exposed to Arg-C proteinase, and Sample 2 is exposed to Asp-N endopeptidase. What will be the sequences of the resulting peptides in each sample at the end of the experiment?2.1.04 Acid-Base Chemistry of PolypeptidesBecause peptides are made from amino acids, and amino acids have (see Lesson 1.3), peptides also have ionizable groups. These ionizable groups behave similarly to those of free amino acids, with some exceptions.pKa Values of N- and C-TerminiThe amino groups of free amino acid backbones have pKa values near 9.6. However, when an amino acid becomes part of a peptide, the pKa of the amino group at its N-terminus tends to decrease to approximately 8.3. Similarly, the pKa of the carboxyl group in a free amino acid backbone is typically near 2.2, but the C-terminus of a peptide has an increased pKa near 3.5 (values specific to a given peptide will usually be given on the exam as needed).Although the ends of a peptide are predominantly charged, the positive charge of the N-terminus cancels the negative charge of the C-terminus at physiological pH. Therefore, just as free amino acid backbones are zwitterionic, so are peptide backbones.Net Charge of a PeptideThe predominant net charge of a peptide can be estimated by evaluating the charges of its backbone and its side chains. Because the backbone is zwitterionic at pH levels found in living cells, the charges of its positive N-terminus and negative C-terminus cancel each other. In addition, the only side chains that predominantly contribute positive charge to a peptide at physiological pH are arginine and lysine, and the only side chains that predominantly contribute negative charge are aspartate and glutamate.As stated in Lesson 1.3, histidine side chains have a pKa of 6. Therefore, most histidine residues are neutral at physiological pH, and only a minority of them contribute a positive charge. However, if the pH drops below 6 (as in ), the predominant form of histidine is positively charged. Similarly, cysteine predominantly contributes a negative charge if the pH increases above 8 (as in some peroxisomes), but only if the cysteine side chain is not involved in a. The pKa of tyrosine is too high to contribute a negative charge to the peptide under conditions that are typically found within cells.Note that estimations of net charge assume that the side chain pKa values are not altered by their proximity to other functional groups in the peptide. In large proteins, pKa values are commonly altered, and the net charge at a given pH must be determined empirically. Chapter 2: Peptides and Proteins44Concept Check 2.3What is the predominant net charge of the peptide ANEQGSKHDIRK at pH 7 and pH 5?Isoelectric Point of a PeptideJust as individual amino acids have isoelectric points (ie, pH value at which the average net charge of a population is 0), so do peptides. The isoelectric point (pI) of a peptide can be estimated in the same way as that of an amino acid---by averaging the pKa below which the peptide is positively charged and the pKa above which the peptide is negatively charged. For short peptides, this can be accomplished relatively easily. Figure 2.9 shows an example estimation of the isoelectric point of the peptide ARE. Chapter 2: Peptides and Proteins45Figure 2.9 Estimation of the isoelectric point of the peptide ARE using pKa values of the free amino acids. However, estimation of pI becomes more difficult as the peptide gets longer, both because there are more ionizable groups to consider and because the pKa values of the groups may change due to interactions with other chemical groups in the peptide.For large peptides and proteins, the pI can be determined empirically using a technique called. This technique uses an electric field to cause the charged peptide or protein of interest to migrate through a gel that contains a pH gradient. Migration continues until the peptide reaches its pI because electrically neutral molecules do not migrate in an electric field. For more information on isoelectric focusing, see Lesson 14.1.Changes to the composition of a peptide alter its pI. In general, addition of an amino acid residue that can carry a positive charge (R, K, and H) increases the pI because a higher pH is needed to fully neutralize the added positive charges. Note that arginine, which has the highest pKa of the three, tends to cause the greatest increase in pI, and histidine causes the smallest increase.Similarly, residues that can carry a negative charge (D, E, C, and Y) decrease the pI because a lower pH is needed to neutralize the negative charges. In this case, aspartate has the lowest pKa and tends to cause the greatest decrease in pI while tyrosine, with a pKa of 10.0, rarely has an impact on pI.Removal of ionizable residues causes the opposite effect; removal of a positively charged residue causes the pI to decrease, whereas removal of a negatively charged residue causes the pI to increase