Marco teórico y teorías científicas relacionadas a la valuación de inventarios: 20

Now let us consider the synthesis of the monomeric units from which biopolymers are made. How can simple one-carbon compounds such as CO₂ and formic acid be incorporated into complex carbon compounds? How can carbon chains grow in length or be shortened? How are branched chains and rings formed?

1. Carbonyl Groups in Chain Formation and Cleavage

Except for some vitamin B₁₂-dependent reactions, the cleavage or formation of carbon–carbon bonds usually depends upon the participation of carbonyl groups. For this reason, carbonyl groups have a central mechanistic role in biosynthesis. The activation of hydrogen atoms β to carbonyl groups permits β con- densations to occur during biosynthesis. Aldol or Claisen condensations require the participation of two carbonyl compounds. Carbonyl compounds are also essential to thiamin diphosphate-dependent condensa- tions and the aldehyde pyridoxal phosphate is needed for most C – C bond cleavage or formation within amino acids.

Because of the importance of carbonyl groups to the mechanism of condensation reactions, much of the assembly of either straight-chain or branched-carbon skeletons takes place between compounds in which the average oxidation state of the carbon atoms is similar to that in carbohydrates (or in formaldehyde, H₂CO). The diversity of chemical reactions possible with compounds at this state of oxidation is a maximum, a fact that may explain why carbohydrates and closely related substances are major biosynthetic precursors and why the average state of oxidation of the carbon in

most living things is similar to that in carbohydrates.174

This fact may also be related to the presumed occurrence of formaldehyde as a principal component of the earth’s atmosphere in the past and to the ability of formaldehyde to condense to form carbohydrates.

In Fig. 17-13 several biochemicals have been arranged according to the oxidation state of carbon. Most of the important biosynthetic intermediates lie within± 2 electrons per carbon atom of the oxidation state of carbohydrates. As the chain length grows, they tend to fall even closer. It is extremely difficult to move through enzymatic processes between 2C, 3C, and 4C compounds (i.e., vertically in Fig. 17-13) except at the oxidation level of carbohydrates or somewhat to its right, at a slightly higher oxidation level. On the other hand, it is often possible to move horizontally with ease using oxidation–reduction reactions. Thus, fatty acids are assembled from acetate units, which lie at the same oxidation state as carbohydrates and, after assembly, are reduced.

Among compounds of the same overall oxidation state, e.g., acetic acid and sugars, the oxidation states of individual carbon atoms can be quite different. Thus, in a sugar every carbon atom can be regarded as immediately derived from formaldehyde, but in acetic acid one end has been oxidized to a carboxyl group and the other has been reduced to a methyl group. Such internal oxidation–reduction reactions play an important role in the chemical manipulations necessary to assemble the carbon skeletons needed by a cell. Decarboxylation is a feature of many biosynthetic routes. Referring again to Fig. 17-13, notice that many of the biosynthetic intermediates such as pyruvate, oxoglutarate, and oxaloacetate are more oxidized than the carbohydrate level. However, their decarboxylation products, which become incorporated into the com- pounds being synthesized, are closer to the oxidation level of carbohydrates.

2. Starting with CO₂

There are three known pathways by which auto- trophic organisms can use CO₂ to synthesize triose phosphates or 3-phosphoglycerate, three-carbon com- pounds from which all other biochemical substances can be formed.175– 177 _{The first of these is the reductive}

tricarboxylic cycle. This is a reversal of the oxidative citric acid cycle in which reduced ferredoxin is used as a reductant in the reaction of Eq. 17-47 to incorporate CO₂ into pyruvate. Succinyl-CoA can react with CO₂ in the same type of reaction to form 2-oxoglutarate, accomplishing the reversal of the only irreversible step in the citric acid cycle. Using these reactions photosyn- thetic bacteria and some anaerobes that can generate a high ratio of reduced to oxidized ferredoxin carry out the reductive tricarboxylic acid cycle. Together with

Acetyl-CoA CO₂

Pyruvate– _{+ CoA}

Fd_red Fd_ox

Figure 17-13 Some biochemical compounds arranged in order of average oxidation state of the carbon atoms and by

carbon-chain lengths. Black horizontal arrows mark some biological interconversions among compounds with the same chain length, while green lines show changes in chain length and are often accompanied by decarboxylation.

HC COH)n O H (H CH2OH (CH2)n H COOH C H O)n ( Carbohydrates 0 -2 +2 +4 -4 -6 CHO COH CH2OH H 0 +4e– CH4

+2e– _–2e– _–4e–

CH3OH H2CO HCOOH CO2 Multicarbon compounds with same oxidation state in all internal carbons CH2OH COH CH2OH H

Acetaldehyde Acetic acid Glycine

+6 2C Dihydroxy- acetone P Serine Glyceraldehyde 4C Alanine Pyruvic acid

Acetone COOH CH2 CH3 COOH CH2 CH2OH COOH CH2 CHO COOH CH2 COOH Malonic semialdehyde n-Butanol Glycerol Oxaloacetic acid COH COH CH3 CH3 H H C COH CH3 CH3 O H C C CH3 CH3 O O CH2 CH2 CHO COOH Acetoin Methylmalonic acid CO2 CO2 COOH C CH2 CH2 COOH O 2-Oxoglutaric acid Oxalosuccinic acid Citric acid CO2 5 & 6C CO2 Total number of electrons from carbohydrate level

3C Glyoxylic acid COOH C CH2OH O COOH HC CH2OH OH COOH HC CH3 COOH CH2 CH2 CH2OH CH2OH CHO CH2OH COOH CH2OH COOH CHO COOH COOH CH2OH CH3 CHO CH3 COOH CH3 COOH CH2NH2 CH3 HCOH CH3 CH3 C CH3 O COOH H2NCH CH3 COOH C CH3 O COOH HCOH HC O COOH C CH2 CH3 COOH CH2 HOCH CH3 COOH HC CH2OH CH3 COOH C CH2 O COOH Malic acid Fumaric acid COOH CH COOH CH Succinic acid COOH CH2 COOH CH2 Acetoacetic acid COOH CH2 CH3 C O Valine COOH C CH3 CH3 CO2 CO2

(17-49)

Eq. 17-47, the cycle provides for the complete synthesis of pyruvate from CO₂.178,179

A quantitatively much more important pathway of CO₂ fixation is the reductive pentose phosphate pathway (ribulose bisphosphate cycle or Calvin– Benson cycle; Fig. 17-14). This sequence of reactions, which takes place in the chloroplasts of green plants and also in many chemiautotrophic bacteria, is essen- tially a way of reversing the oxidative pentose phos- phate pathway (Fig. 17-8). The latter accomplishes the complete oxidation of glucose or of glucose 1-phosphate by NADP+ _{(Eq. 17-48):}

Glucose 1-P2– _{+ ATP}4 – _{+ 8 H}

2O + 12 NADP +→

6 CO₂ + 12 NADPH + ADP3– _{+ 2HPO}

42 – + 13 H +

∆G’ (pH 7) = –299 kJ mol –1

It would be almost impossible for a green plant to fix CO₂ using photochemically generated NADPH by an exact reversal of Eq. 17-48 because of the high positive Gibbs energy change. To solve this thermo- dynamic problem the reductive pentose phosphate pathway has been modified in a way that couples ATP cleavage to the synthesis.

The reductive carboxylation system is shown within the green shaded box of Fig. 17-14. Ribulose 5-phosphate is the starting compound and in the first step one molecule of ATP is expended to form ribulose 1,5-bisphosphate. The latter is carboxylated and cleaved to two molecules of 3-phosphoglycerate. This reaction was discussed in Chapter 13. The reductive step (step c) of the system employs NADPH together with ATP. Except for the use of the NADP system instead of the NAD system, it is exactly the reverse of the glyceraldehyde phosphate dehydrogenase reaction of glycolysis. Looking at the first three steps of Fig. 17-14 it is clear that in the reductive pentose phosphate pathway three molecules of ATP are utilized for each CO₂ incorporated. On the other hand, in the oxidative direction no ATP is generated by the operation of the pentose phosphate pathway.

The reactions enclosed within the shaded box of Fig. 17-14 do not give the whole story about the cou- pling mechanism. A phospho group was transferred from ATP in step a and to complete the hydrolysis it must be removed in some future step. This is indicated in a general way in Fig. 17-14 by the reaction steps

d, e, and f. Step f represents the action of specific phos-

phatases that remove phospho groups from the seven- carbon sedoheptulose bisphosphate and from fructose bisphosphate. In either case the resulting ketose monophosphate reacts with an aldose (via transketolase, step g) to regenerate ribulose 5-phosphate, the CO₂ acceptor. The overall reductive pentose phosphate cycle (Fig. 17-14B) is easy to understand as a reversal of the oxidative pentose phosphate pathway in which the oxidative decarboxylation system of Eq. 17-12 is

replaced by the reductive carboxylation system of Fig. 17-14A. The scheme as written in Fig. 17-14B shows the incorporation of three molecules of CO₂. The reductive carboxylation system operates three times with a net production of one molecule of triose phos- phate. As with other biosynthetic cycles, any amount of any of the intermediate metabolites may be with- drawn into various biosynthetic pathways without disruption of the flow through the cycle.

The overall reaction of carbon dioxide reduction in the Calvin–Benson cycle (Fig. 17-14) becomes

6 CO₂+ 12 NADPH + 18 ATP4 –_{+ 11 H} 2O→

glucose-1-P2- _{+ 12 NADP}+ _{+ 18 ADP}3 –

17 HPO₄2 – _{+ 6 H} +

The Gibbs energy change ∆G’ (pH 7) is now – 357 kJ mol–1 _{instead of the + 299 kJ mol} –1 _{required to reverse}

the reaction of Eq. 17-48.

The third pathway for reduction of CO₂ to acetyl- CoA is utilized by acetogenic bacteria, by methanogens, and probably by sulfate-reducing bacteria.179– 181

This acetyl-CoA pathway (or Wood – Ljungdahl pathway) involves reduction by H₂ of one of the two molecules of CO₂ to the methyl group of methyl- tetrahydromethanopterin in methanogens and of methyltetra-hydrofolate in acetogens. The pathway utilized by methanogens is illustrated in Fig. 15-22.182– 184 _{A similar process utilizing H}

2 as the reductant is

employed by acetogens.179,185– 188a _{In both cases a}

methyl corrinoid is formed and its methyl group is condensed with a molecule of carbon monoxide bound to a copper ion in a Ni–Cu cluster.189a,b _{The resulting}

acetyl group is transferred to a molecule of coenzyme A as illustrated in Eq. 16-52.189 _{The bound CO is}

formed by reduction of CO₂, again using H₂ as the reductant.190 _{The overall reaction for acetyl-CoA}

synthesis is given by Eq. 17-50. Conversion of acetyl- CoA to pyruvate via Eq. 17-47 leads into the glucogen- ic pathway. (17-48) CH3 Pterin CH3 Co (Corrin) CH3 C O S CoA + 3 H2O CO2 6-Electron reduction (Fig. 15-22) CoA-SH CO– CH3 C O S CoA Carbon monoxide dehydrogenase CO CO2 2e 2 CO2 + 4 H2 + CoASH (Eq. 16-52) Overall: – Ni Cu (17-50)

An alternative pathway by which some acetogenic bacteria form acetate is via reversal of the glycine decar- boxylase reaction of Fig. 15-20. Methylene-THF is formed by reduction of CO₂, and together with NH₃ and CO₂ a lipoamide group of the enzyme and PLP forms glycine. The latter reacts with a second methylene- THF to form serine, which can be deaminated to pyru- vate and assimilated. Methanogens may use similar pathways but ones that involve methanopterin (Fig. 15-17).191

3. Biosynthesis from Other Single-Carbon Compounds

Various bacteria and fungi are able to subsist on such one-carbon compounds as methane, methanol, methylamine, formaldehyde, and formate.192 – 197 _Energy

is obtained by oxidation to CO₂. Methylotrophic bacteria initiate oxidation of methane by hydroxylation (Chapter 18) and dehydrogenate the resulting methanol or exogenous methanol using the PPQ cofactor (Eq. 15-51).198 _{Further dehydrogenation to formate and of}

formate to CO₂ via formate dehydrogenase (Eq. 16-63) completes the process. Some methylotrophic bacteria incorporate CO₂ for biosynthetic purposes via the ribulose bisphosphate (Calvin–Benson) cycle but many use pathways that begin with formaldehyde (or methylene-THF). Others employ variations of the reductive pentose phosphate pathway to convert formaldehyde to triose phosphate. In one of these, the ribulose monophosphate cycle or Quayle cycle,192,193

ribulose 5-P undergoes an aldol condensation with formaldehyde to give a 3-oxo-hexulose 6-phosphate (Eq. 17-51, step a). The latter is isomerized to fructose 6-P (Eq. 17-51, step b). If this equation is applied to the

BOX 17-E 14_{C AND THE CALVIN – BENSON CYCLE}

The chemical nature of photosynthesis had intrigued chemists for decades but little was learned about the details until radioactive 14_{C became avail-}

able. Discovered in 1940 by Ruben and Kamen, the isotope was available in quantity by 1946 as a prod- uct of nuclear reactors. Initial studies of photosyn- thesis had been conducted by Ruben and Kamen using11_{C but} 14_{C made rapid progress possible. In}

1946 Melvin Calvin and Andrew A. Benson began their studies that elucidated the mechanism of incor- poration of CO₂ into organic materials.

A key development was two-dimensional paper chromatography with radioautography (Box 3-C). A suspension of the alga Chlorella (Fig. 1-11) was allowed to photosynthesize in air. At a certain time, a portion of H14_CO

3 was injected into the system,

and after a few seconds of photosynthesis with 14_C

present the suspension of algae was run into hot methanol to denature proteins and to stop the reac- tion. The soluble materials extracted from the algal cells were concentrated and chromatographed; radioautographs were then prepared. It was found that after 10 s of photosynthesis in the presence of

14_CO

2, the algae contained a dozen or more 14C

labeled compounds. These included malic acid, aspartic acid, phosphoenolpyruvate, alanine, triose phosphates, and other sugar phosphates and diphos- phates. However, during the first five seconds a single

compound, 3-phosphoglycerate, contained most of the radioactivity.a,b _{This finding suggested that a two-}

carbon regenerating substrate might be carboxylated by14_CO

2 to phosphoglycerate. Search for this two-

carbon compound was unsuccessful, but Benson, in Calvin’s laboratory, soon identified ribulose

a _{Benson, A. A., Bassham, J. A., Calvin, M., Goodale, T. C., Haas,}

V. A., and Stepka, W. (1950) J. Am. Chem. Soc. 72, 1710 – 1718

b _{Benson, A. A. (1951) J. Am. Chem. Soc. 73, 2971 – 2972}

c _{Benson, A. A., Kawaguchi, S., Hayes, P., and Calvin, M. (1952)}