Chemical energy: chemical energy can be found in the form of high energy molecules. These molecules are required for energetically unfavorable reactions. Furthermore, the hydrolysis of these compounds drives a reaction forward. High energy molecules, such as ATP, have three phosphates. Often, the terminal phosphate is split off during hydrolysis and transferred to another molecule.
Coenzymes or cofactors: cofactors are molecules that assist in chemical reactions. These may be metal ions, vitamin derivatives such as NADH and acetyl CoA, or non-vitamin derivatives such as ATP. In the case of NADH, the molecule transfers a hydrogen, whereas acetyl CoA transfers an acetyl group, and ATP transfers a phosphate.
In the simplest sense, the reactions that occur in biosynthesis have the following format:
Some variations of this basic equation which will be discussed later in more detail are:
Simple compounds which are converted into other compounds, usually as part of a multiple step reaction pathway. Two examples of this type of reaction occur during the formation of nucleic acids and the charging of tRNA prior to translation. For some of these steps, chemical energy is required:
Simple compounds that are converted into other compounds with the assistance of cofactors. For example, the synthesis of phospholipids requires acetyl CoA, while the synthesis of another membrane component, sphingolipids, requires NADH and FADH for the formation the sphingosine backbone. The general equation for these examples is:
Simple compounds that join together to create a macromolecule. For example, fatty acids join together to form phospholipids. In turn, phospholipids and cholesterol interact noncovalently in order to form the lipid bilayer. This reaction may be depicted as follows:
Lipid membrane bilayer
Many intricate macromolecules are synthesized in a pattern of simple, repeated structures. For example, the simplest structures of lipids are fatty acids. Fatty acids are hydrocarbon derivatives; they contain a carboxyl group "head" and a hydrocarbon chain "tail". These fatty acids create larger components, which in turn incorporate noncovalent interactions to form the lipid bilayer.
Fatty acid chains are found in two major components of membrane lipids: phospholipids and sphingolipids. A third major membrane component, cholesterol, does not contain these fatty acid units.
The foundation of all biomembranes consists of a bilayer structure of phospholipids. The phospholipid molecule is amphipathic; it contains a hydrophilic polar head and a hydrophobic nonpolar tail. The phospholipid heads interact with each other and aqueous media, while the hydrocarbon tails orient themselves in the center, away from water. These latter interactions drive the bilayer structure that acts as a barrier for ions and molecules.
The pathway starts with glycerol 3-phosphate, which gets converted to lysophosphatidate via the addition of a fatty acid chain provided by acyl coenzyme A. Then, lysophosphatidate is converted to phosphatidate via the addition of another fatty acid chain contributed by a second acyl CoA; all of these steps are catalyzed by the glycerol phosphate acyltransferase enzyme. Phospholipid synthesis continues in the endoplasmic reticulum, and the biosynthesis pathway diverges depending on the components of the particular phospholipid.
Sphingolipids are formed from ceramides that consist of a fatty acid chain attached to the amino group of a sphingosine backbone. These ceramides are synthesized from the acylation of sphingosine. The biosynthetic pathway for sphingosine is found below:
As the image denotes, during sphingosine synthesis, palmitoyl CoA and serine undergo a condensation reaction which results in the formation of dehydrosphingosine. This product is then reduced to form dihydrospingosine, which is converted to sphingosine via the oxidation reaction by FAD.
Cholesterol is synthesized from acetyl CoA. The pathway is shown below:
More generally, this synthesis occurs in three stages, with the first stage taking place in the cytoplasm and the second and third stages occurring in the endoplasmic reticulum. The stages are as follows:
The DNA nucleotides adenosine and guanosine consist of a purine base attached to a ribose sugar with a glycosidic bond. In the case of RNA nucleotides deoxyadenosine and deoxyguanosine, the purine bases are attached to a deoxyribose sugar with a glycosidic bond. The purine bases on DNA and RNA nucleotides are synthesized in a twelve-step reaction mechanism present in most single-celled organisms. Higher eukaryotes employ a similar reaction mechanism in ten reaction steps. Purine bases are synthesized by converting phosphoribosyl pyrophosphate (PRPP) to inosine monophosphate (IMP), which is the first key intermediate in purine base biosynthesis. Further enzymatic modification of IMP produces the adenosine and guanosine bases of nucleotides.
N5-CAIR synthetase transfers a carboxyl group, forming the intermediate N5-carboxyaminoimidazole ribonucleotide (N5-CAIR).
N5-CAIR mutase rearranges the carboxyl functional group and transfers it onto the imidazole ring, forming carboxyamino- imidazole ribonucleotide (CAIR). The two step mechanism of CAIR formation from AIR is mostly found in single celled organisms. Higher eukaryotes contain the enzyme AIR carboxylase, which transfers a carboxyl group directly to AIR imidazole ring, forming CAIR.
After the uridine nucleotide base is synthesized, the other bases, cytosine and thymine are synthesized. Cytosine biosynthesis is a two-step reaction which involves the conversion of UMP to UTP. Phosphate addition to UMP is catalyzed by a kinase enzyme. The enzyme CTP synthase catalyzes the next reaction step: the conversion of UTP to CTP by transferring an amino group from glutamine to uridine; this forms the cytosine base of CTP. The mechanism, which depicts the reaction UTP + ATP + glutamine CTP + ADP + glutamate, is below:
Cytosine is a nucleotide that is present in both DNA and RNA. However, uracil is only found in RNA. Therefore, after UTP is synthesized, it is must be converted into a deoxy form to be incorporated into DNA. This conversion involves the enzyme ribonucleoside triphosphate reductase. This reaction that removes the 2'-OH of the ribose sugar to generate deoxyribose is not affected by the bases attached to the sugar. This non-specificity allows ribonucleoside triphosphate reductase to convert all nucleotide triphosphates to deoxyribonucleotide by a similar mechanism.
In contrast to uracil, thymine bases are found mostly in DNA, not RNA. Cells do not normally contain thymine bases that are linked to ribose sugars in RNA, thus indicating that cells only synthesize deoxyribose-linked thymine. The enzyme thymidylate synthetase is responsible for synthesizing thymine residues from dUMP to dTMP. This reaction transfers a methyl group onto the uracil base of dUMP to generate dTMP. The thymidylate synthase reaction, dUMP + 5,10-methylenetetrahydrofolate dTMP + dihydrofolate, is shown to the right.
As DNA polymerase moves in a 3' to 5' direction along the template strand, it synthesizes a new strand in the 5' to 3' direction
Although there are differences between eukaryotic and prokaryotic DNA synthesis, the following section denotes key characteristics of DNA replication shared by both organisms.
DNA synthesis is initiated by the RNA polymeraseprimase, which makes an RNA primer with a free 3'OH. This primer is attached to the single-stranded DNA template, and DNA polymerase elongates the chain by incorporating nucleotides; DNA polymerase also proofreads the newly synthesized DNA strand.
During the polymerization reaction catalyzed by DNA polymerase, a nucleophilic attack occurs by the 3'OH of the growing chain on the innermost phosphorus atom of a deoxynucleoside triphosphate; this yields the formation of a phosphodiester bridge that attaches a new nucleotide and releases pyrophosphate.
Two types of strands are created simultaneously during replication: the leading strand, which is synthesized continuously and grows towards the replication fork, and the lagging strand, which is made discontinuously in Okazaki fragments and grows away from the replication fork. Okazaki fragments are covalently joined by DNA ligase to form a continuous strand.
Then, to complete DNA replication, RNA primers are removed, and the resulting gaps are replaced with DNA and joined via DNA ligase.
The general structure of the standard amino acids includes a primary amino group, a carboxyl group and the functional group attached to the ?-carbon. The different amino acids are identified by the functional group. As a result of the three different groups attached to the ?-carbon, amino acids are asymmetrical molecules. For all standard amino acids, except glycine, the ?-carbon is a chiral center. In the case of glycine, the ?-carbon has two hydrogen atoms, thus adding symmetry to this molecule. With the exception of proline, all of the amino acids found in life have the L-isoform conformation. Proline has a functional group on the ?-carbon that forms a ring with the amino group.
One major step in amino acid biosynthesis involves incorporating a nitrogen group onto the ?-carbon. In cells, there are two major pathways of incorporating nitrogen groups. One pathway involves the enzyme glutamine oxoglutarate aminotransferase (GOGAT) which removes the amide amino group of glutamine and transfers it onto 2-oxoglutarate, producing two glutamate molecules. In this catalysis reaction, glutamine serves as the nitrogen source. An image illustrating this reaction is found to the right.
The other pathway for incorporating nitrogen onto the ?-carbon of amino acids involves the enzyme glutamate dehydrogenase (GDH). GDH is able to transfer ammonia onto 2-oxoglutarate and form glutamate. Furthermore, the enzyme glutamine synthetase (GS) is able to transfer ammonia onto glutamate and synthesize glutamine, replenishing glutamine.
The biosynthesis of glutamate and glutamine is a key step in the nitrogen assimilation discussed above. The enzymes GOGAT and GDH catalyze the nitrogen assimilation reactions.
In bacteria, the enzyme glutamate 5-kinase initiates the biosynthesis of proline by transferring a phosphate group from ATP onto glutamate. The next reaction is catalyzed by the enzyme pyrroline-5-carboxylate synthase (P5CS), which catalyzes the reduction of the ?-carboxyl group of L-glutamate 5-phosphate. This results in the formation of glutamate semialdehyde, which spontaneously cyclizes to pyrroline-5-carboxylate. Pyrroline-5-carboxylate is further reduced by the enzyme pyrroline-5-carboxylate reductase (P5CR) to yield a proline amino acid.
There are two distinct lysine biosynthetic pathways: the diaminopimelic acid pathway and the ?-aminoadipate pathway. The most common of the two synthetic pathways is the diaminopimelic acid pathway; it consists of several enzymatic reactions that add carbon groups to aspartate to yield lysine:
Aspartate kinase initiates the diaminopimelic acid pathway by phosphorylating aspartate and producing aspartyl phosphate.
4-hydroxy-tetrahydrodipicolinate reductase catalyzes the reduction of (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate by NADPH to yield ?'-piperideine-2,6-dicarboxylate (2,3,4,5-tetrahydrodipicolinate) and H2O.
The serine family of amino acid includes: serine, cysteine, and glycine. Most microorganisms and plants obtain the sulfur for synthesizing methionine from the amino acid cysteine. Furthermore, the conversion of serine to glycine provides the carbons needed for the biosynthesis of the methionine and histidine.
The aspartate family of amino acids includes: threonine, lysine, methionine, isoleucine, and aspartate. Lysine and isoleucine are considered part of the aspartate family even though part of their carbon skeleton is derived from pyruvate. In the case of methionine, the methyl carbon is derived from serine and the sulfur group, but in most organisms, it is derived from cysteine.
The biosynthesis of aspartate is a one step reaction that is catalyzed by a single enzyme. The enzyme aspartate aminotransferase catalyzes the transfer of an amino group from aspartate onto ?-ketoglutarate to yield glutamate and oxaloacetate. Asparagine is synthesized by an ATP-dependent addition of an amino group onto aspartate; asparagine synthetase catalyzes the addition of nitrogen from glutamine or soluble ammonia to aspartate to yield asparagine.
The diaminopimelic acid lysine biosynthetic pathway
The diaminopimelic acid biosynthetic pathway of lysine belongs to the aspartate family of amino acids. This pathway involves nine enzyme-catalyzed reactions that convert aspartate to lysine.
Aspartate kinase catalyzes the initial step in the diaminopimelic acid pathway by transferring a phosphoryl from ATP onto the carboxylate group of aspartate, which yields aspartyl-?-phosphate.
Siaminopimelate decarboxylase catalyzes the final step in lysine biosynthesis that removes the carbon dioxide group from meso-diaminopimelic acid to yield L-lysine.
The tRNA anticodon interacts with the mRNA codon in order to bind an amino acid to growing polypeptide chain.
The process of tRNA charging
Protein synthesis occurs via a process called translation. During translation, genetic material called mRNA is read by ribosomes to generate a protein polypeptide chain. This process requires transfer RNA (tRNA) which serves as an adaptor by binding amino acids on one end and interacting with mRNA at the other end; the latter pairing between the tRNA and mRNA ensures that the correct amino acid is added to the chain. Protein synthesis occurs in three phases: initiation, elongation, and termination.Prokaryotic translation differs from eukaryotic translation; however, this section will mostly focus on the commonalities between the two organisms.
Before translation can begin, the process of binding a specific amino acid to its corresponding tRNA must occur. This reaction, called tRNA charging, is catalyzed by aminoacyl tRNA synthetase. A specific tRNA synthetase is responsible for recognizing and charging a particular amino acid. Furthermore, this enzyme has special discriminator regions to ensure the correct binding between tRNA and its cognate amino acid. The first step for joining an amino acid to its corresponding tRNA is the formation of aminoacyl-AMP:
This is followed by the transfer of the aminoacyl group from aminoacyl-AMP to a tRNA molecule. The resulting molecule is aminoacyl-tRNA:
The combination of these two steps, both of which are catalyzed by aminoacyl tRNA synthetase, produces a charged tRNA that is ready to add amino acids to the growing polypeptide chain.
In addition to binding an amino acid, tRNA has a three nucleotide unit called an anticodon that base pairs with specific nucleotide triplets on the mRNA called codons; codons encode a specific amino acid. This interaction is possible thanks to the ribosome, which serves as the site for protein synthesis. The ribosome possesses three tRNA binding sites: the aminoacyl site (A site), the peptidyl site (P site), and the exit site (E site).
There are numerous codons within an mRNA transcript, and it is very common for an amino acid to be specified by more than one codon; this phenomenon is called degeneracy. In all, there are 64 codons, 61 of each code for one of the 20 amino acids, while the remaining codons specify chain termination.
Translation in steps
As previously mentioned, translation occurs in three phases: initiation, elongation, and termination.
Step 1: Initiation
The completion of the initiation phase is dependent on the following three events:
1. The recruitment of the ribosome to mRNA
2. The binding of a charged initiator tRNA into the P site of the ribosome
3. The proper alignment of the ribosome with mRNA's start codon
Step 2: Elongation
Following initiation, the polypeptide chain is extended via anticodon:codon interactions, with the ribosome adding amino acids to the polypeptide chain one at a time. The following steps must occur to ensure the correct addition of amino acids:
1. The binding of the correct tRNA into the A site of the ribosome
2. The formation of a peptide bond between the tRNA in the A site and the polypeptide chain attached to the tRNA in the P site
3. Translocation or advancement of the tRNA-mRNA complex by three nucleotides
Translocation "kicks off" the tRNA at the E site and shifts the tRNA from the A site into the P site, leaving the A site free for an incoming tRNA to add another amino acid.
Step 3: Termination
The last stage of translation occurs when a stop codon enters the A site. Then, the following steps occur:
1. The recognition of codons by release factors, which causes the hydrolysis of the polypeptide chain from the tRNA located in the P site
Errors in biosynthetic pathways can have deleterious consequences including the malformation of macromolecules or the underproduction of functional molecules. Below are examples that illustrate the disruptions that occur due to these inefficiencies.
Familial hypercholesterolemia: this disorder is characterized by the absence of functional receptors for LDL. Deficiencies in the formation of LDL receptors may cause faulty receptors which disrupt the endocytic pathway, inhibiting the entry of LDL into the liver and other cells. This causes a buildup of LDL in the blood plasma, which results in atherosclerotic plaques that narrow arteries and increase the risk of heart attacks.
Severe combined immunodeficiency (SCID): SCID is characterized by a loss of T cells. Shortage of these immune system components increases the susceptibility to infectious agents because the affected individuals cannot develop immunological memory. This immunological disorder results from a deficiency in adenosine deanimase activity, which causes a buildup of dATP. These dATP molecules then inhibit ribonucleotide reductase, which prevents of DNA synthesis.
^Mueller, EJ; Meyer, E; Rudolph, J; Davisson, VJ; Stubbe, J (Mar 1, 1994). "N5-carboxyaminoimidazole ribonucleotide: evidence for a new intermediate and two new enzymatic activities in the de novo purine biosynthetic pathway of Escherichia coli". Biochemistry. 33 (8): 2269-78. doi:10.1021/bi00174a038. PMID8117684.
^Firestine, SM; Poon, SW; Mueller, EJ; Stubbe, J; Davisson, VJ (Oct 4, 1994). "Reactions catalyzed by 5-aminoimidazole ribonucleotide carboxylases from Escherichia coli and Gallus gallus: a case for divergent catalytic mechanisms". Biochemistry. 33 (39): 11927-34. doi:10.1021/bi00205a031. PMID7918411.
^Broach, edited by Jeffrey N. Strathern, Elizabeth W. Jones, James R. (1981). The Molecular biology of the yeast Saccharomyces. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory. ISBN978-0879691394.CS1 maint: extra text: authors list (link)
^ abcdGeer, Gerald Karp ; responsible for the revision of chapter 15 Peter van der (2004). Cell and molecular biology : concepts and experiments (4th ed., Wiley International ed.). New York: J. Wiley & Sons. ISBN978-0471656654.
^PETERKOFSKY, B; GILVARG, C (May 1961). "N-Succinyl-L-diaminopimelic-glutamic transaminase". The Journal of Biological Chemistry. 236: 1432-8. PMID13734750.
^KINDLER, SH; GILVARG, C (December 1960). "N-Succinyl-L-2,6-diaminopimelic acid deacylase". The Journal of Biological Chemistry. 235: 3532-5. PMID13756049.
^Born, TL; Blanchard, JS (October 1999). "Structure/function studies on enzymes in the diaminopimelate pathway of bacterial cell wall biosynthesis". Current Opinion in Chemical Biology. 3 (5): 607-13. doi:10.1016/s1367-5931(99)00016-2. PMID10508663.
^Wang, W; Kim, R; Jancarik, J; Yokota, H; Kim, SH (Jan 10, 2001). "Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution". Structure. 9 (1): 65-71. doi:10.1016/s0969-2126(00)00558-x. PMID11342136.
^Monschau, N; Stahmann, KP; Sahm, H; McNeil, JB; Bognar, AL (May 1, 1997). "Identification of Saccharomyces cerevisiae GLY1 as a threonine aldolase: a key enzyme in glycine biosynthesis". FEMS Microbiology Letters. 150 (1): 55-60. doi:10.1111/j.1574-6968.1997.tb10349.x. PMID9163906.
^Pye, VE; Tingey, AP; Robson, RL; Moody, PC (Sep 24, 2004). "The structure and mechanism of serine acetyltransferase from Escherichia coli". The Journal of Biological Chemistry. 279 (39): 40729-36. doi:10.1074/jbc.M403751200. PMID15231846.
^McPhalen, CA; Vincent, MG; Picot, D; Jansonius, JN; Lesk, AM; Chothia, C (Sep 5, 1992). "Domain closure in mitochondrial aspartate aminotransferase". Journal of Molecular Biology. 227 (1): 197-213. doi:10.1016/0022-2836(92)90691-C. PMID1522585.
^Larsen, TM; Boehlein, SK; Schuster, SM; Richards, NG; Thoden, JB; Holden, HM; Rayment, I (Dec 7, 1999). "Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product". Biochemistry. 38 (49): 16146-57. CiteSeerX10.1.1.453.5998. doi:10.1021/bi9915768. PMID10587437.
^Velasco, AM; Leguina, JI; Lazcano, A (October 2002). "Molecular evolution of the lysine biosynthetic pathways". Journal of Molecular Evolution. 55 (4): 445-59. doi:10.1007/s00239-002-2340-2. PMID12355264.
^Kotaka, M; Ren, J; Lockyer, M; Hawkins, AR; Stammers, DK (Oct 20, 2006). "Structures of R- and T-state Escherichia coli aspartokinase III. Mechanisms of the allosteric transition and inhibition by lysine". The Journal of Biological Chemistry. 281 (42): 31544-52. doi:10.1074/jbc.M605886200. PMID16905770.
^Hadfield, A; Kryger, G; Ouyang, J; Petsko, GA; Ringe, D; Viola, R (Jun 18, 1999). "Structure of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli, a key enzyme in the aspartate family of amino acid biosynthesis". Journal of Molecular Biology. 289 (4): 991-1002. doi:10.1006/jmbi.1999.2828. PMID10369777.
^Mirwaldt, C; Korndörfer, I; Huber, R (Feb 10, 1995). "The crystal structure of dihydrodipicolinate synthase from Escherichia coli at 2.5 A resolution". Journal of Molecular Biology. 246 (1): 227-39. doi:10.1006/jmbi.1994.0078. PMID7853400.
^Cirilli, M; Zheng, R; Scapin, G; Blanchard, JS (Sep 16, 2003). "The three-dimensional structures of the Mycobacterium tuberculosis dihydrodipicolinate reductase-NADH-2,6-PDC and -NADPH-2,6-PDC complexes. Structural and mutagenic analysis of relaxed nucleotide specificity". Biochemistry. 42 (36): 10644-50. doi:10.1021/bi030044v. PMID12962488.
^Pillai, B; Cherney, M; Diaper, CM; Sutherland, A; Blanchard, JS; Vederas, JC; James, MN (Nov 23, 2007). "Dynamics of catalysis revealed from the crystal structures of mutants of diaminopimelate epimerase". Biochemical and Biophysical Research Communications. 363 (3): 547-53. doi:10.1016/j.bbrc.2007.09.012. PMID17889830.
^Gokulan, K; Rupp, B; Pavelka MS, Jr; Jacobs WR, Jr; Sacchettini, JC (May 16, 2003). "Crystal structure of Mycobacterium tuberculosis diaminopimelate decarboxylase, an essential enzyme in bacterial lysine biosynthesis". The Journal of Biological Chemistry. 278 (20): 18588-96. doi:10.1074/jbc.M301549200. PMID12637582.
^ abcWalport, Ken Murphy, Paul Travers, Mark (2011). Janeway's Immunobiology (8. ed.). Oxford: Taylor & Francis. ISBN978-0815342434.
^ abHughes, edited by Donald C. Lo, Robert E. (2010). Neurobiology of Huntington's disease : applications to drug discovery (2nd ed.). Boca Raton: CRC Press/Taylor & Francis Group. ISBN978-0849390005.CS1 maint: extra text: authors list (link)