| Chem 431 |
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Fall 2001 |
| Lecture Notes:: 14 December |
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| PREVIOUS |
Note that two ATP's are required to synthesize carbamyl phosphate, the first to activate bicarbonate, the second to provide the activated phosphate on the carbamyl-P itself. Carbamyl-P is itself a high-energy compound with a mixed anhydride bond much like we saw in 1,3-bis PGA. The carbamyl group is then transferred onto ornithine which acts as a carrier for the growing urea molecule. Addition of the aspartate nitrogen requires two additional ATP equivalents to drive the condensation to argininosuccinate. Lysis results in the formation of a fumerate and arginine, which is then hydrolyzed to give our product, Urea, and regenerate the ornithine carrier.
Note a total of four ATP equivalents has been consumed. How many ATP equivalents are then required to convert the nitrogen of two amino acids into urea? The flow diagram supplied below may help in this calculation:
To solve this problem we can first note the four ATP equivalents consumed in the biosynthesis of one urea from two amino acids. But the fumerate produced must be taken back to regenerate the oxalacetate used to pick up the nitrogen from one amino acid):
This will provide an NADH via malate DH. This is equivalent to 2.5 ATP's, so we have -4 + 2.5 = -1.5. Next, while the second nitrogen enters via two transaminations through aspartate:
we also have to produce the ammonia from the first amino acid via glutamate using Glutamate DH,
This also produces an NADH, providing another 2.5 ATP's. thus the final tally is -4 + 2.5 + 2.5 = +1 ATP to produce one urea from two amino acid nitrogens. Of course for mammals this does not take into account the physiological costs of excreting the urea, which can be significant.
The enzymes involved in the conversion of amino acid nitrogen into urea occur in the cytosol and in the mitosol and involve three different catalytic systems: the Urea Cycle, The TCA cycle, and a transamination cycle, you might call them "Kreb's Tricycle." These interactions are shown in the figure in your packet (Kreb's Tricycle). Note the involvement of two antiports to move intermediates across the inner mitochondrial membrane: the malate: aspartate antiport, and the citrulline:ornithine antiport. Note also that nitrogen is incorporated in the mitosol (Transamination, Carbamoyl-P synthesis), whereas the final product, urea is released in the cytosol.
Most amino acids are metabolized predominantly in the liver, but "alkyl" (branched chain) amino acids (val, leu, & ilu) are preferentially metabolized by skeletal muscle, whereas the "acyl" (asp, asn, glu, & gln) are metabolized in the intestinal mucosa. As an example let's look at the metabolism of protein after a one day fast (tissues need fuel and glucose). If we start with 1,000 mM of amino acids (equivalent to a "steak dinner" of 530 g of lean raw meat. (Data from McGilvery, Biochemistry: a Functional Approach, 1979, based on idealized calculations for amino acid distributions.):
Note that most of the alanine coming to the liver is from the muscle and intestine, produced from amino acid nitrogen and pyruvate to keep serum ammonia concentrations down. One of the most striking aspects of this chart is the difference in tissue usage of ATP (note that all calculations are based on 3 ATP/NADH and 2 ATP/FADH2). Both muscle and intestine derive significant energy the amino acids they breakdown. On the other hand, nearly all of the energy from the amino acids metabolized in the liver goes into glucose (very little ATP is made by the liver), which would go largely to the CNS (15,048 ATP @ 38 ATP's/Glucose, based on 3 ATP/NADH and 2 ATP/FADH2 used in the other calculations. If we use the values of 2.5 ATP/NADH and 1.5 ATP/FADH2, then we get 12,672 ATP @ 32 ATP/glucose)!
Much of the nitrogen is carried between the tissues and the liver by the Alanine Cycle:
A similar, but more complex cycle involves glutamate/glutamine taking nitrogen from the muscle to the intestine where the glutamine is catabolized and the nitrogen goes to alanine which then goes to the liver. Glucose can then go to the muscle and be converted to glutamate via Glycolysis and the TCA cycle. We will not look at this more complex system.
The catabolic breakdown of most of the amino acids is summarized in the Main Routes of Amino Acid Catabolism diagram in your packet. A couple of overview comments. First, quite a number of aa catabolic pathways have irreversible steps, as symbolized by the heavy arrows in the diagram. These amino acids will be essential (that is must be provided by the diet). Generally we find that amino acids are essential in mammals (cannot be synthesized) if they are only needed to make protein. Non-essential aa's, such as serine, are biosynthesized by us because they have important roles in intermediary metabolism, not because they are needed to make protein. Second, amino acids can be categorized as being glucogenic (can be used in Gluconeogenesis) or ketogenic (cannot be used in Gluconeogenesis). Most aa's can be at least partially used in glucose synthesis. But ilu is only partially glucogenic (note some goes directly to acetyl-CoA), while leu and lys are fully ketogenic.
We will begin by looking at the catabolism of amino acids by groups: 3-C (feed into pyruvate), 4-C (feed into oxalacetate), and 5-C (feed into glutamate).
3-C aa's: Ser and ala are converted in single step processes to pyruvate. Cys is converted after first oxidizing and removing sulfur as sulfate.
4-C aa's: Asn is hydrolyzed in one step to aspartate, which in turn is transaminated in one step to oxalacetate. Threonine feeds into the TCA cycle through succinyl-CoA instead of oxalacetate. Thr is first deaminated via a dehydratase as seen earlier, then decarboxylated by Pyruvate DH Complex to give propionyl-CoA, which is then transformed via a series of steps to give succinyl-CoA.
Propionyl-CoA metabolism: propionyl-CoA is an intermediate in the catabolism of a number of amino acids, as well as in the breakdown of odd-chain fatty acids. Propionyl-CoA (3-C) enters the TCA Cycle at succinyl-CoA (4-C), thus another carbon must be added to bring it into mainstream metabolism. A biotin-dependent carboxylase adds carbon dioxide at the cost of one ATP to give D-methylmalonyl-CoA. D-methylmalonyl-CoA is then racemized to L-methylmalonyl-CoA. Methylmalonyl-CoA is a branched-chain, whereas succinyl-CoA is straight-chain: the carboxyl group and a hydrogen must be exchanged. This exchange requires C-C bond-breaking and making, a process apparently involving a Co-C bond intermediate. The cobalamin cofactor derived from Vit B12 is used in catalyzing this reaction {overhead}.
5-C aa's: Five aa's feed into glutamate which in turns feeds into the TCA cycle at 2-oxo-glutarate. His is first deaminated, then the ring is opened and the formamino group is then donated to the one-carbon pool (see later). Two of these reactions are irreversible so his is essential. Proline is first oxidized and then hydrolyzed to open the ring and give glutamaldehyde which is oxidized to give glutamate. Note that the glutamaldehyde tends to spontaneously refold to the ring, which can then be reduced to synthesize proline. It is thus not essential. Gln is hydrolyzed in one step to glutamate. Arginine is hydrolyzed to ornithine by arginase from the urea cycle. Ornithine is then transaminated to glutamaldehyde as seen with proline. Arginine is essential for infants because the arginase removes essentially all of the arg made in the urea cycle, and glutamaldehydes tendency to cyclize means it cannot be effectively synthesized from glutamate. (Bacteria use a blocking group to stop cyclization at this stage.)
Branched chain amino acids: val, leu, and ilu. The metabolism of each of these three amino acids begins with the same theme: transaminase; DH Complex; b-oxidation. Due to the irreversible nature of the DH Complex all three are essential. In the case of ilu this pattern leads to propionyl-CoA without modification. Val goes through the first two steps of b-oxidation after which its structure dictates different reactions to reach propionyl-CoA. With leu the b-oxidation is interrupted after the first step, at which point carbon dioxide is added to give 3-methyl-3-hydroxy-glutaryl-CoA the same HMG seen in ketone body synthesis. Its remaining catabolism is a reversal of ketone body synthesis.
Lysine: Note the unusual "transamination" of the epsilon amino group where lysine is first reduced using NADPH and condensed with 2-oxo-glutarate to give L-saccharopine. Saccharopine is then split and oxidized using NAD+ to give glutamate and "lysine aldehyde." The aldehyde is then oxidized again and the resulting 2-aminoadipate now follows the branched chain pattern: transaminase, DH Complex, and beta-oxidation to give acetoacetyl-CoA and finally two acetyl-CoA's.
Tyrosine and Phenyalanine: The last two amino acids on the diagram are broken into two parts: half feeds into the TCA cycle at fumerate (glucogenic), and the other half goes to acetoacetate (ketogenic). Phe is first hydroxylated using molecular oxygen and the cofactor tetrahydrobiopteran to give tyr. Tyrosine is thus only an essential aa if insufficient phe is present in the diet to synthesize it. Tyr is next transaminated followed by a couple of oxidations of the benzene ring using molecular oxygen and involving iron as a cofactor. These reactions open the ring, which is then hydrolyzed to give fumerate and acetoacetate.
 Pathway Diagrams |
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