Carboxylation enzymes

The chemistry of a fourth coenzyme was at least partially elucidated in the period under discussion. F. Lynen and coworkers treated P-methylcrotonyl coenzyme A (CoA) carboxylase with bicarbonate labelled with 14C, and discovered that one atom of radiocarbon was incorporated per molecule of enzyme. They postulated that an intermediate was formed between the enzyme and C02, in which the biotin of the enzyme had become car-boxylated. The carboxylated enzyme could transfer its radiolabelled carbon dioxide to methylcrotonyl CoA more interestingly, they found that the enzyme-COz compound would also transfer radiolabelled carbon dioxide to free biotin. The resulting compound, carboxybiotin [4], was quite unstable, but could be stabilized by treatment with diazomethane to yield the methyl ester of N-carboxymethylbiotin (7) (Lynen et al., 1959). The identification of this radiolabelled compound demonstrated that the unstable material is N-carboxybiotin itself, which readily decarboxylates esterification prevents this reaction, and allows the isolation and identification of the product. Lynen et al. then postulated that the structure of the enzyme-C02 compound was essentially the same as that of the product they had isolated from the reaction with free biotin, but where the carbon dioxide was inserted into the bound biotin of the enzyme (Lynen et al., 1961). Although these discoveries still leave significant questions to be answered as to the detailed mechanism of the carboxylation reactions in which biotin participates as coenzyme, they provide a start toward elucidating the way in which the coenzyme functions. 

A second approach is to use [lsO]bicarbonate and to follow the incorporation of lsO into a carboxylated substrate. If C02 is the primary substrate only two labeled oxygen atoms enter the compound, whereas if HC03 is the reactant three are incorporated.287 A third technique is measurement of the rate of incorporation of C02 or bicarbonate in the carboxylated product. Over a short interval of time, e.g., 1 min, different kinetics will be observed for the incorporation of C02 and of bicarbonate.288 Using these methods, it was established that the product formed in Eq. 13-46 and the reactant in Eq. 13-47 is C02. However, the carboxylation enzymes considered in the next section use bicarbonate as the substrate. 

In this cycle, one molecule of acetyl-CoA is formed from two molecules of bicarbonate (Figure 3.5). The key carboxylating enzyme is the bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase. In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step of fatty acid biosynthesis. However, Archaea do not contain fatty acids in their lipids, and acetyl-CoA carboxylase cannot serve as the key enzyme of fatty acid synthesis rather, it is responsible for autotrophy. 

This cycle resembles the 3-hydroxypropionate/4-hydroxybutyrate cycle, but with pyruvate ferredoxin oxidoreductase (pyruvate synthase) and phosphoenolpyruvate (PEP) carboxylase as the carboxylating enzymes (Figure 3.6). 

Today the metabolic network of the central metabolism of C. glutamicum involving glycolysis, pentose phosphate pathway (PPP), TCA cycle as well as anaplerotic and gluconeogenetic reactions is well known (Fig. 1). Different enzymes are involved in the interconversion of carbon between TCA cycle (malate/oxaloacetate) and glycolysis (pyruvate/phosphoenolpyruvate). For anaplerotic replenishment of the TCA cycle, C. glutamicum exhibits pyruvate carboxylase [20] and phosphoenol-pyruvate (PEP) carboxylase as carboxylating enzymes. Malic enzyme [21] and PEP carboxykinase [22,23] catalyze decarboxylation reactions from the TCA cycle... 

Figure 8-11. Schematic cross section near the periphery of a mesophyll cell (Fig. 1-1), indicating the sequential anatomical components across which CO2 diffuses from the intercellular air spaces to the carboxylation enzymes in the chloroplast stroma.

The answer is b. (Murray, pp 627-661. Scriver, pp 3897-3964. Sack, pp 121-138. Wilson, pp 287-320.) The vitamin biotin is the cofactor required by carboxylating enzymes such as acetyl CoA, pyruvate, and propionyl CoA carboxylases. The fixation of CO2 by these biotin-dependent enzymes occurs in two stages. In the first, bicarbonate ion reacts with adenosine triphosphate (ATP) and the biotin carrier protein moiety of the enzyme in the second, the active CO2 reacts with the substrate—e.g., acetyl CoA. 

The increase in 02 and concomitant decrease in C02 caused by the evolution of oxygenic autotrophs on the Earth, has resulted in an undersaturation of the main (andancient) carboxylating enzyme, ribulose-l,5-bisphosphate carboxylase, responsible for the first step in carbon fixation—the dark reaction of photosynthesis. To palliate this difficulty, a number of species of marine phytoplankton have evolved carbon concentrating mechanisms that all involve some forms of... 

The earlier flux analyses based on positional enrichment patterns did not succeed in resolving the two C3-carboxylating enzymes phosphoeno/pyruvate carboxylase (PEPCx) and pyruvate carboxylase (PyrCx) because the carbon routes in both reactions to oxaloacetate are identical and because no differences... 

The recovery of assimilation rate included both an increase in the efficiency of the carboxylation processes and the level of carbon reduction cycling. This was probably due to increasing reductant production as the light reactions recovered, which would both induce carboxylation enzymes and allow greater levels of carbon cycling. 

Another issue associated with the pH is carbon dioxide fixation. As outlined above, probably all succinate production pathways are critically dependent on the net fixation of carbon dioxide by C3 carboxylation reactions. Enzymes responsible for this conversion are PEP carboxylase, PEPCK, pyruvate carboxylase, and malic enzyme. Studies on the active species of CO2 used by these enzymes indicated that PEP carboxylase and pyruvate carboxylase use bicarbonate as substrate, whereas PEP carboxykinase and malic enzyme use CO2 (for references see Bott and Thauer, 1989). Under acidic conditions, the equilibrium of the reaction CO2 (aq) -I- H2O 5 HCO3 -l-H+(p a = 6. 33 at 30°C) is strongly shifted to carbon dioxide, which may positively or negatively impact carbon dioxide fixation, depending on the carboxylating enzymes present. 

Since its demonstration in photosynthetic organisms, the carboxylation enzyme has been demonstrated in E, coli and Thiobacillus. The physiological significance of this enzyme in nonphotosynthetic organisms is not clear, but its occurrence emphasizes that the carbon metabolism of photosynthesis is an enzymatic process distinct from the photochemical reaction. 

P-enolpyruvate carboxylase was first observed and measured in spinach leaf extracts by Bandurski and Greiner (1953), and was quickly shown to be an important carboxylating enzyme in succulent plants (e.g.. Walker, 1957). Since its discovery and implication in Crassulacean Acid Metabolism, it has been studied... 

Osmond, C.B., Greenway, H. Salt responses of carboxylation enzymes from species differing in salt tolerance. Plant Physiol. 49,260-262 (1972)... 

Biotin is the prosthetic group of carboxylating enzymes, such as acetyl-CoA-carboxylase, pyruvate carboxylase and propionyl-CoA-carboxylase, and therefore plays an important role in fatty acid biosynthesis and in gluconeo-genesis. The carboxyl group of biotin forms an amide bond with the e-amino group of a lysine residue of the particular enzyme protein. Only the (3aS, 4S, 6aR) compound, D-(-i-)-biotin, is biologically active ... 

Atmospheric CO2 may be fixed through the Calvin-Benson-Bassham-cycle [13, 23-25]. The key carboxylating enzyme, D-ribulose-l,5-bisphosphate carboxylase/ oxygenase (RubisCO) [26], binds CO2 onto a pentose derivative, ribulose-1,5-bisphosphate. It has been estimated that about 4 x 10 g (40 Mt) of RubisCO exists in the biosphere, which would correspond to an intangible 5 kg of RubisCO per person on Earth [27]. 

These biochemical transformations occur on a multienzyme complex composed of at least three dissimilar proteins biotin carrier protein (MW = 22,000), biotin carboxylase (MW = 100,000) and biotin transferase (MW = 90,000). Each partial reaction is specifically catalyzed at a separate subsite and the biotin is covalently attached to the carrier protein through an amide linkage to a lysyl a-amino group of the carrier protein (338, 339). In 1971, J. Moss and M. D. Lane, from Johns Hopkins University proposed a model for acetyl-CoA carboxylase of E, coli where the essential role of biotin in catalysis is to transfer the fixed CO2, or carboxyl, back and forth between two subsites. Consequently, reactions catalyzed by a biotin-dependent carboxylase proceed though a carboxylated enzyme complex intermediate in which the covalently bound biotinyl prosthetic poup acts as a mobile carboxyl carrier between remote catalytic sites (Fig. 7.13). 

Tietz and Ochoa could detect no biotin in their enzyme preparation although in biotin deficiency carboxylation of propionyl CoA is greatly diminyied. After it was established that biotin is the coenzyme of the carboxylation enzyme of jS-methylcrotonyl CoA lOJf) it was soon shown that this is also true for the propionyl CoA carboxylase 105a, lOSb). 

DTT, and EDTA, (4) pepstatin and iodoacetate. The first tube would be expected to reflect the full proteolytic activity of all protein-ases except for metalloproteases the second would be expected to permit only the expression of non-thiol proteases the third would be expected to show no cathepsin D activity, and the fourth would be expected to block all thiol and carboxyl enzymes. Calculations based on these four values were used to estimate the results in Figure 2. 

The carboxylation enzyme is activated by sulfhydryl compounds such as cysteine and glutathione and is inhibited by agents which react with sulfhydryl groups. It requires Mg++ or other divalent ions, such as Ni++ or Co++, and is inhibited by relatively low concentrations of phosphate or arsenate. The enzyme is specific for RuDP and has a high affinity for this substrate, and is useful for the quantitative determination of this substance. PGA formed in the reaction can be estimated by several enzymic methods (386). 

The formation of an enediol intermediate is supported by the observation that one of the carbon-bound protons of RuDP was labilized by the carboxylation enzyme and would exchange with tritium-labeled water (J. Hurwitz and W. B. Jakoby, unpublished observation). PGA formed in the presence of COj and tritium water also contained tritium. 

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