Pyruvate, amino acid synthesis

Matrix of the mitochondrion This gel-like solution in the interior of mitochondria is fifty percent protein. These molecules include the enzymes responsible for the oxidation of pyruvate, amino acids, fatty acids (by p-oxidation), and those of the tricarboxylic acid (TCA) cycle. The synthesis of urea and heme occur partially in the matrix of mitochondria. In addition, the matrix contains NAD+and FAD (the oxidized forms of the two coenzymes that are required as hydrogen acceptors) and ADP and Pj, which are used to produce ATP. [Note The matrix also contains mitochondrial RNA and DNA (mtRNA and mtDNA) and mitochondrial ribosomes.]... 

In cases where the natural amino acid side chains of enzymes are insufficient to carry out a desired reaction, the enzyme frequently uses coenzymes. A coenzyme is bound by the enzyme along with the substrate, and the enzyme catalyses the bimolecular reaction between the coenzyme and the substrate (cf. Section 2.6.3). A simple model for a-amino acid synthesis by transamination was developed by substituting /I-cyclodextrin with pyridoxamine. Pyridoxamine is able to carry out the transformation of a-keto acids to a-amino acids even without the presence of the cyclodextrin, but with the cyclodextrin cavity as well, the enzyme model proves to be more selective and transaminates substrates with aryl rings bound strongly by the cyclodextrin much more rapidly than those having little affinity for the cyclodextrin. Thus (p-le/f-butylphenyl) pyruvic acid and phenylpyruvic acid are transaminated respectively 15 000 and 100 times faster then pyruvic acid itself, to give (p-le/f-butylphenyl) alanine and phenylalanine (Scheme 12.5). 

On the basis of the similarities in their synthetic pathways, the amino acids can be grouped into six families glutamate, serine, aspartate, pyruvate, the aromatics, and histidine. The amino acids in each family are ultimately derived from one precursor molecule. In the discussions of amino acid synthesis that follow, the intimate relationship between amino acid metabolism and several other metabolic pathways is apparent. Amino acid biosynthesis is outlined in Figure 14.4. 

Another hypothesis of aromatic amino acid synthesis, based on the distribution of the label in tyrosine of yeast grown on radioactive pyruvate or acetate, is that it involves the cyclic condensation of two unsym-metric 4-carbon acids, e.g., oxalacetate. The side chain of tyrosine appears to be formed from pyruvate as an intact 3-carbon unit. 

Pyruvate carboxylase catalyses the conversion of pyruvate to oxaloacetate using ATP and CO2. This is an important reaction both for gluconeogenesis (to bypass the pyruvate kinase reaction) and also for the normal function of the citric acid cyde. For the citric acid cycle to begin, one molecule each of oxaloacetate and acetyl CoA is required. If there is a shortage of oxaloacetate, the balance is restored by the action of pyruvate carboxylase. If, for example, oxaloacetate has been removed from the cycle to enter the amino acid synthesis pathways, it can simply be regenerated fiom pyruvate (31b). 

As noted above the operation of the TCA cycle results in the conversion of acetyl-CoA to carbon dioxide and reduced co-enzymes, the oxidation of which may be coupled to the synthesis of ATP (see terminal oxidation below). However the TCA cycle must not be thought of simply as an incinerator—it is also important as a means whereby acetyl-Co A from a variety of sources is converted to compounds involved in a number of biosynthetic pathways. For example oxoglutarate is involved in amino acid synthesis and succinyl-CoA is a precursor of porphyrins. These and other intermediates are withdrawn from the cycle—however, a moment s thought will indicate that the carbon withdrawn must be replaced if the rate of utilization of acetyl-Co A by the cycle is not to be reduced. The reactions shown below result in the replenishment of the cycle with malic or oxaloacetic acid from phosphoenolpyruvate (PEP) or pyruvate and are sometimes referred to as anaplerotic reactions. 

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

The acetyl-CoA derived from amino acid degradation is normally insufficient for fatty acid biosynthesis, and the acetyl-CoA produced by pyruvate dehydrogenase and by fatty acid oxidation cannot cross the mitochondrial membrane to participate directly in fatty acid synthesis. Instead, acetyl-CoA is linked with oxaloacetate to form citrate, which is transported from the mitochondrial matrix to the cytosol (Figure 25.1). Here it can be converted back into acetyl-CoA and oxaloacetate by ATP-citrate lyase. In this manner, mitochondrial acetyl-CoA becomes the substrate for cytosolic fatty acid synthesis. (Oxaloacetate returns to the mitochondria in the form of either pyruvate or malate, which is then reconverted to acetyl-CoA and oxaloacetate, respectively.)... 

FIGURE 25.1 The citrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing equivalents (electrons) for fatty acid synthesis. The shuttle collects carbon substrates, primarily from glycolysis but also from fatty acid oxidation and amino acid catabolism. Most of the reducing equivalents are glycolytic in origin. Pathways that provide carbon for fatty acid synthesis are shown in blue pathways that supply electrons for fatty acid synthesis are shown in red. 

Yet a third method for the synthesis of a-amino acids is by reductive amination of an a-keto acid with ammonia and a reducing agent. Alanine, for instance, is prepared by treatment of pyruvic acid with ammonia in the presence of NaBH As described in Section 24.6, the reaction proceeds through formation of an intermediate imine that is then reduced. 

Deoxy-araWno-heptulosonic acid 7-phosphate (10) is a metabolic intermediate before shikimic acid in the biosynthetic pathway to aromatic amino-acids in bacteria and plants. While (10) is formed enzymically from erythrose 4-phosphate (11) and phosphoenol pyruvate, a one-step chemical synthesis from (11) and oxalacetate has now been published.36 The synthesis takes place at room temperature and neutral pH... 

As we have seen, normally pyruvate would be the substrate for pyruvate dehydrogenase complex to form acetyl-CoA, but during fasting in the absence of glucose, acetyl -CoA for the TCA cycle is derived from fatty acid (3-oxidation (see Section 7.5.2) so pyruvate is diverted into oxaloacetate by the enzyme pyruvate carboxylase. Thus any amino acids whose carbon skeletons can be converted into pyruvate, OAA or another substrate of the TCA cycle, can be used for glucose synthesis. 

This enzyme [EC 4.1.99.1], also known as L-tryptophan indole-lyase, catalyzes the hydrolysis of L-tryptophan to generate indole, pyruvate, and ammonia. The reaction requires pyridoxal phosphate and potassium ions. The enzyme can also catalyze the synthesis of tryptophan from indole and serine as well as catalyze 2,3-elimination and j8-replacement reactions of some indole-substituted tryptophan analogs of L-cysteine, L-serine, and other 3-substituted amino acids. 

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