Modification of Polymers
There are three chemical prob- lems associated with the assembly of a protein, nucleic acid, or other biopolymer. The first is to overcome
thermodynamic barriers. The second
is to control the rate of synthesis, and the third is to establish the pattern or
sequence in which the monomer units are linked together. Let us look briefly
at how these three problems are dealt with by living cells.
1. Peptides and Proteins Activation of amino acids for incorporation into oligopeptides and proteins can occur via two routes of acyl activation. In the first of these an acyl phosphate (or acyl adenylate) is formed and reacts with an amino group to form a peptide linkage (Eq. 13-4). The tripeptide glutathione is formed in two steps of this type (Box 11-B). In the second method of activation aminoacyl
O C S CoA CH2 H3C Propionyl-CoA HCO3– Biotin ATP O C S ACP C H3C –OOC H C O H3C S E Acetyl-enzyme H3C C C C S ACP O O H3C H Methylmalonyl-ACP CO2 O C H3C CH2 C S CoA O H3C C O S CoA 2 Acetyl-CoA CH2 C C –OOC S O CoA OH H3C HMG-CoA CH2 C CH2 –OOC CH 2OH OH H3C Mevalonate C CH2 CH2 O P P Isopentenyl pyrophosphate H2C H3C H3C C COO– O C COO– CH3 O CO2 H3C C C COO– O HO CH3 H3C C COO– O H H3C Valine (Isoleucine) α-Acetolactate
1. Starter piece for branched-chain fatty acids
2. Polyisoprenoid compounds
3. Branched-chain amino acids
Figure 17-19 Biosynthetic origins of three five-carbon branched structural
adenylates are formed. They transfer their activated aminoacyl groups onto specific tRNA molecules dur- ing synthesis of proteins (Eq. 17-36). In other cases activated aminoacyl groups are transferred onto – SH groups to form intermediate thioesters. An example is the synthesis of the antibiotic gramicidin S formed by Bacillus brevis. The antibiotic is a cyclic decapeptide with the following five-amino-acid sequence repeated twice in the ringlike molecule211:
(–d-Phe –l-Pro – l-Val – l-Orn – l-Leu – )2
The soluble enzyme system responsible for its synthesis contains a large 280-kDa protein that not only activates the amino acids as aminoacyl adenylates and transfers them to thiol groups of 4'-phosphopantetheine groups covalently attached to the enzyme but also serves as a template for joining the amino acids in proper sequence.211 – 214 Four amino acids —proline,
valine, ornithine (Orn), and leucine—are all bound. A second enzyme (of mass 100 kDa) is needed for acti- vation of phenylalanine. It is apparently the activated phenylalanine (which at some point in the process is isomerized from L- to D-phenylalanine) that initiates
polymer formation in a manner analogous to that of fatty acid elongation (Fig. 17-12). Initiation occurs when the amino group of the activated phenylalanine (on the second enzyme) attacks the acyl group of the aminoacyl thioester by which the activated proline is held. Next, the freed imino group of proline attacks the activated valine, etc., to form the pentapeptide. Then two pentapeptides are joined and cyclized to give the antibiotic. The sequence is absolutely specific, and it is remarkable that this relatively small enzyme system is able to carry out each step in the proper sequence. Many other peptide antibiotics, such as the bacitracins, tyrocidines,215 and enniatins, are synthesized
in a similar way,213,216,217 as are depsipeptides and the
immunosuppresant cyclosporin. A virtually identical pattern is observed for formation of polyketides,218,219
whose chemistry is considered in Chapter 21.
While peptide antibiotics are synthesized according to enzyme-controlled polymerization patterns, both proteins and nucleic acids are made by template mechanisms. The sequence of their monomer units is determined by genetically encoded information. A key reaction in the formation of proteins is the transfer of activated aminoacyl groups to molecules of tRNA (Eq. 17-36). The tRNAs act as carriers or adapters as explained in detail in Chapter 29. Each aminoacyl- tRNA synthetase must recognize the correct tRNA and attach the correct amino acid to it. The tRNA then carries the activated amino acid to a ribosome, where it is placed, at the correct moment, in the active site. Peptidyltransferase, using a transacylation reaction, in an insertion mechanism transfers the C terminus of the growing peptide chain onto the amino group of
the new amino acid to give a tRNA-bound peptide one unit longer than before.
2. Polysaccharides
Incorporation of a sugar monomer into a polysac- charide also involves cleavage of two high-energy phosphate linkages of ATP. However, the activation process has its own distinctive pattern (Eq. 17-56). Usually a sugar is first phosphorylated by a kinase or a kinase plus a phosphomutase (Eq. 17-56, step a). Then a nucleoside triphosphate (NuTP) reacts under the influence of a second enzyme with elimination of pyrophosphate and formation of a glycopyranosyl ester of the nucleoside diphosphate, more often known as a sugar nucleotide (Eq. 17-56, step b). The inorganic pyrophosphate is hydrolyzed by pyro- phosphatase while the sugar nucleotide donates the activated glycosyl group for polymerization (Eq. 17-56, step c). In this step the glycosyl group is transferred with displacement of the nucleoside diphosphate. Thus, the overall process involves first the cleavage of ATP to ADP and Pi, and then the cleavage of a nucleo- side triphosphate to a nucleoside diphosphate plus Pi. The nucleoside triphosphate in Eq. 17-56, step b is sometimes ATP, in which case the overall result is the splitting of two molecules of ATP to ADP. However, as detailed in Chapter 20, the whole series of nucleotide “handles” serve to carry various activated glycosyl units.
What determines the pattern of incorporation of sugar units into polysaccharides? Homopolysaccharides, like cellulose and the linear amylose form of starch, contain only one monosaccharide component in only one type of linkage. A single synthetase enzyme can add unit after unit of an activated sugar (UDP glucose or other sugar nucleotide) to the growing end. However, at least two enzymes are needed to assemble a branched molecule such as that of the glycogen molecule. One is the synthetase; the second is a branching enzyme, a transglycosylase. After the chain ends attain a length of about ten monosaccharide units the branching enzyme attacks a glycosidic linkage somewhere in the chain. Acting much like a hydrolase, it forms a glyco- syl enzyme (or a stabilized carbocation) intermediate. The enzyme does not release the severed chain frag- ment but transfers it to another nearby site on the branched polymer. In the synthesis of glycogen, the chain fragment is joined to a free 6-hydroxyl group of the glycogen, creating a new branch attached by an α-1,6-linkage.
Other carbohydrate polymers consist of repeating oligosaccharide units. Thus, in hyaluronan units of glucuronic acid and N-acetyl-D-glucosamine alternate
(Fig. 4-11). The “O antigens” of bacterial cell coats (p. 180) contain repeating subunits made up of a “block” of four or five different sugars. In these and
many other cases the pattern of polymerization is established by the specificities of individual enzymes. An enzyme capable of joining an activated glucosyl unit to a growing polysaccharide will do so only if the proper structure has been built up to that point. In cases where a block of sugar units is transferred it is usually inserted at the nonreducing end of the polymer, which may be covalently attached to a protein. Notice that the insertion mode of chain growth exists for lipids, polysaccharides, and proteins.
3. Nucleic Acids
The activated nucleotides are the nucleoside 5'-triphosphates. The ribonucleotides ATP, GTP, UTP, and CTP are needed for RNA synthesis and the 2'-deoxyribonucleotide triphosphates, dATP, dTTP, dGTP, and dCTP for DNA synthesis. In every case, the addition of activated monomer units to a growing polynucleotide chain is catalyzed by an enzyme that
binds to the template nucleic acid. The choice of the proper nucleotide unit to place next in the growing strand is determined by the nucleotide already in place in the complementary strand, a matter that is dealt with in Chapters 27 and 28. The chemistry is a simple displacement of pyrophosphate (Eq. 17-57). The 3'-hydroxyl of the polynucleotide attacks the phos- phorus atom of the activated nucleoside triphosphate. Thus, nucleotide chains always grow from the 5' end, with
new units being added at the 3' end.
4. Phospholipids and Phosphate – Sugar Alcohol Polymers
Choline and ethanolamine are activated in much the same way as are sugars. For example, choline can be phosphorylated using ATP (Eq. 17-58, step a) and the phosphocholine formed can be further converted (Eq. 17-58, step b) to cytidine diphosphate choline. Phosphocholine is transferred from the latter onto a suitable acceptor to form the final product (Eq. 17-58, step c). The polymerization pattern differs from that for polysaccharide synthesis. When the sugar nucleo- tides react, the entire nucleoside diphosphate is eliminated (Eq. 17-56), but CDP-choline and CDP- ethanolamine react with elimination of CMP (Eq. 17-58, step c), leaving one phospho group in the final product. The same thing is true in the synthesis of the bacterial teichoic acids (Chapter 8). Either CDP- glycerol or CDP-ribitol is formed first and polymeriza- tion takes place with elimination of CMP to form the alternating phosphate–sugar alcohol polymer.220
5. Irreversible Modification and Catabolism of Polymers
While polymers are being synthesized continuously by cells, they are also being modified and torn down. Nothing within a cell is static. As discussed in Chapters
O O PO32– ATP ADP Kinase a b Nucleotide triphosphate PPi H2O 2 Pi Glycopyranosyl ester of nucleoside diphosphate, e.g., UDP-glucose O O H H
One or more steps
O
O Acceptor
Linked glycosyl unit
Nucleoside diphosphate c O O P O O O H Acceptor end of polysaccharide O O Nucleoside – P O O – (17-56) PPi HO Nucleotide O P O O O– P O O O– P O– O OH HO Nu O P O O O– 3' 5' Polynucleotide 3' O Polynucleotide 5' 5' H H2O 2 Pi (17-57)
10 and 29, everything turns over at a slower or faster rate. Hydrolases attack all of the polymers of which cells are composed, and active catabolic reactions de- grade the monomers formed. Membrane surfaces are also altered, for example, by hydroxylation and glyco- sylation of both glycoproteins and lipid head groups. It is impossible to list all of the known modification reactions of biopolymers. They include hydrolysis, methylation, acylation, isopentenylation, phospho- rylation, sulfation, and hydroxylation. Precursor molecules are cut and trimmed and often modified further to form functional proteins or nucleic acids. Phosphotransferase reactions splice RNA transcripts to form mRNA and a host of alterations convert pre- cursors into mature tRNA molecules (Chapter 28). Even DNA, which remains relatively unaltered, undergoes a barrage of chemical attacks. Only because of the presence of an array of repair enzymes (Chapter 27) does our DNA remain nearly unchanged so that faithful copies can be provided to each cell in our bodies and can be passed on to new generations.