conversion of glyceraldehyde-3-phosphate to glycerate-1,3-bisphosphate, catalysed by glyceraldehyde-3-phosphate dehydrogenase using Pi and NAD+ (step 6), ATP is formed by the dephosphorylation of glycerate-1,3-bisphosphate (step 7). The second substrate-level phosphorylation is catalysed by pyruvate kinase (step 10) and ATP is produced from the phosphate group of phosphoenolpyruvate. The hexose molecule is split into two three-carbon molecules, via an aldolase reaction (step 4). Hexose is converted to fructose-1,6-bisphosphate by hexokinase (step 1) and phosphofructokinase (step 3a), in the process consuming 2 mol and producing 4 mol of ATP in steps 7 and 10. Thus, there is net production of 2 mol of ATP. The ATP-consuming and ATP-producing reactions of glycolysis are illustrated in Figure 3.4. If the conversion is performed by hexokinase (step 1) and pyrophosphate (PPi)-dependent phosphofructokinase (step 3b), net production of ATP is 3 mol from 1 mol of hexose. Two mol of NADH produced by glycolysis can be used in oxidative phosphorylation in mitochondria to generate more ATP (Zeeman 2015).
A further substrate-level phosphorylation occurs in the TCA cycle. In the matrix of the mitochondria, a substrate-level phosphorylation occurs at the succinate-CoA ligase reaction step (step 6 in Fig. 3.2). In contrast to animals, in plants ATP-specific succinyl-CoA synthetase (EC 6.2.1.5), but not GTP-succinyl-CoA synthetase (EC 6.2.1.4), acts as an enzyme in the TCA cycle (see Figure 3.4). This enzyme produces ATP from ADP accompanied by degradation of succinyl-CoA (Table 3.1 and Figure 3.4c). For further details of this topic, readers can refer to comprehensive plant biochemistry text books such as Bowsher et al. (2012) and Buchanan et al. (2015).
3.4.3 Nucleoside-Diphosphate Kinase
Using cellular ATP, purine and pyrimidine nucleoside triphosphate are produced by nucleoside-diphosphate kinase (EC 2.7.4.6). The enzyme catalyses the reaction: nucleoside diphosphate + ATP ↔ nucleoside triphosphate + ADP. Substrate specificity is broad. All purine and pyrimidine nucleoside diphosphates can act as acceptors, while not only ATP, but also any ribonucleoside- and deoxyribonucleoside triphosphate, can act as a donor. This enzyme contributes principally to the formation of nucleoside triphosphates, such as GTP, uridine-triphosphate (UTP) and cytidine-triphosphate (CTP), from the respective nucleoside-diphosphates. Nucleoside-diphosphate kinase occurs ubiquitously in animals, plants, and bacteria. Recent molecular genetic studies suggest that nucleoside-diphosphate kinases are major players in the synthesis of macromolecules since they provide the triphosphates used for cell anabolism, including nucleic acid synthesis, CTP for lipid synthesis, UTP for polysaccharide synthesis, and GTP for protein elongation, signal transduction, and microtubule polymerization. Five nucleoside-diphosphate kinase genes have been detected in Arabidopsis thaliana and rice (Oryza sativa). Cytosolic (type I) and chloroplast (type II) enzymes are involved in metabolism, growth, and stress responses and in photosynthetic development and oxidative stress management, respectively. Type III enzymes are located in mitochondria and chloroplasts and are involved principally in energy metabolism. The subcellular localization and precise function of the novel type IV enzyme has not as yet been determined (Dorion and Rivoal 2015).
Figure 3.3 Glycolysis in plants. (1) Hexokinase; (2) phosphoglucoisomerase; (3a) phosphofructokinase; (3b) pyrophosphate dependent-fructose-6-phosphate 1-phosphotransferase; (4) aldolase; (5) triose phosphate isomerase; (6) glyceraldehyde-3-phosphate dehydrogenase; (7) phosphoglycerate kinase; (8) phosphoglycerate mutase; (9) enolase; (10) pyruvate kinase; (11) amylase etc.; (12) starch phosphorylase; (13) invertase; (14) sucrose synthase; (15) phosphoglucomutase; (16) UDP-glucose pyrophosphorylase; (17) fructokinase. Note: Fructose* produced from sucrose (reaction 13) is converted to fructose-6-P (reaction 17).
Figure 3.4 Reactions involved in the substrate level ATP production and consumption. (1) succinyl CoA synthetase; (2) hexokinase; (3) phosphofructokinase; (4) phosphoglycerate kinase; (5) pyruvate kinase.
3.5 Biosynthesis of Deoxyribonucleotides
Reduction of the ribose moiety of the ribonucleotide occurs at the ribonucleoside diphosphate level by ribonucleotide reductase (EC 1.17.4.1). The reaction is:
dNDP is phosphorylated to dNTP and used for replication and repair in DNA biosynthesis. It has been shown that ribonucleotide reductase, comprising two large (R1) and two small (R2) subunits, catalyses a rate-limiting step in the production of deoxyribonucleotides required for DNA synthesis. The large subunit (R1) contains the allosteric regulatory sites, while the small subunit (R2) encompasses a binuclear iron centre and a tyrosyl free radical (Elledge et al. 1992). In mammals, defective ribonucleotide reductase often leads to cell cycle arrest, growth retardation, and apoptosis, whereas abnormally increased levels result in higher mutation rates. Similar phenomena have been demonstrated in mutants of A. thaliana. The results suggest that ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and general development in plants (Wang and Liu 2006). The pathway for thymidine nucleotides, which are required for DNA synthesis, is described in Chapter 10
3.6 Nucleic Acid Biosynthesis
DNA polymerases (DNA-directed DNA polymerase, EC 2.7.7.7) synthesize DNA from deoxyribonucleotides. These enzymes are essential for DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule. During this process, DNA polymerase ‘reads’ the existing DNA strand to create two new strands that match the existing one.
These enzymes catalyse the following chemical reaction:
DNA polymerase adds nucleotides to the 3′ end of a DNA strand, one nucleotide at a time. At least 15 classes of DNA polymerase have been identified in animals and terminal deoxyribonucleotidyl transferases. Based on their properties, the polymerases (α to σ) are classified into four families, A, B, X, and Y (Burgers et al. 2001). There are few papers on plant DNA polymerases although there is one which reports that plant DNA polymerase-γ is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol β and terminal deoxyribonucleotidyl transferase (Uchiyama et al. 2004).
Biosynthesis of RNA is catalysed by RNA polymerase (DNA-directed RNA polymerase, EC 2.7.7.6). The reaction is:
RNA polymerase, locally, opens the double-stranded DNA (usually about four turns of the double helix) so that one strand of the exposed nucleotides can be used as a template for the synthesis of RNA, namely transcription. A transcription factor and its associated transcription mediator complex must be attached to a DNA binding site, a promoter region, before RNA polymerase can initiate the DNA unwinding at that position. RNA polymerase has intrinsic helicase activity, therefore, no additional enzyme is required to unwind the DNA, in contrast to DNA polymerase. RNA polymerase, not only initiates RNA transcription, it also guides the nucleotides into position,
