|Lecture Notes: 30 April||
Protein catabolism and anabolism are often out of sync - either no additional protein is needed or the amino acid composition of the synthesized proteins is not identical to the protein being hydrolyzed. Neither protein nor amino acids are stored as such. Thus organisms must frequently degrade excess amino acids. Two paths may be available: deaminate the unneeded amino acids and breakdown the carbon skeletons for energy or for storage as fat or carbohydrate and eliminate the nitrogen; or the nitrogen may be transferred to another carbon backbone to make a needed amino acid.
Nitrogen can be eliminated in a variety of forms, depending on the physiological conditions experienced by the organism:
Amino Acid Deamination: For any of these products nitrogen must first be removed from the amino acids. Ammonia is often an intermediate if not the final product in all of these organisms. Most ammonia results from the deamination of a single amino acid: glutamate, via the Glutamate dehydrogenase reaction:
This enzyme is somewhat unusual in that it can use either NADH or NADPH. Glutamate DH is activated by ADP and inhibited by GTP in vitro so they may regulate it in vivo. Of course these nucleotides would not provide a highly sensitive regulation, activity would only loosely follow energy charge.
How are the other amino acids deaminated? Most are transaminated, transferring their N to make glutamate:
amino acid + 2-oxoglutarate 2-oxoacid + glutamate
Transamination of Amino Acids: There are three main transaminases or Amino transferases, all requiring Pyridoxal-P as a cofactor:
The various aminotransferases in the liver all funnel excess N to glutamate and aspartate. Glutamate can then be deaminated by Glutamate DH to give ammonia, contributing up to 1/2 of the N in urea. Aspartate provides N directly to urea synthesis, contributing 1/2 of urea N.
Let's look at the aminotransferase reaction and the cofactor pyridoxal-phosphate. Pyridoxal-P (PLP) is derived from vitamin B6 (pyridoxine) via phosphorylation. Vitamin B6 is thus essential for protein metabolism.
Pyridoxal-P is used to form Schiff bases with its various substrates, but in this case the nitrogen is provided by the substrate. PLP acts as an electrophilic catalyst; since none of the amino acids are electrophilic, cofactors must be provided when electrophilic covalent catalysis is needed.
As we saw before the transaminases catalyze symmetrical reactions:
An amino acid reacts with a keto acid to give an amino acid and a keto acid. Thus one might expect a symmetrical mechanism, which is in fact the case. In the diagram below only the first half of the reaction mechanism is shown. The second half simply "reflects" the first half. It involves substituting the second keto acid in the right-hand step and reversing the process to take the second amino acid off in the left-hand step to complete the reaction (change the R s to R' s in the reverse steps):
Note that the PLP starts out covalently linked to a lysine-N, which is displaced by the reactant amino acid. During the remainder of the reaction the PLP is held non-covalently in the active site. In the final step the lysine amino group on the enzyme displaces the product amino acid nitrogen to release the free amino acid.
Chemically the reactant amino acid is oxidized (aldimine-ketimine shift in the mechanism) to release the keto acid product. The keto acid reactant is then reduced (ketimine-aldimine shift) to release the amino acid product. As might be expected the enzyme exhibits Ping-Pong Bi Bi kinetics:
Pyridoxal-P catalyzes a variety of other reactions involving amino acids involving destabilization of bonds to the alpha-carbon:
In addition, Pyridoxal-P catalyzes the direct deamination of serine and threonine via the destabilization of the side chain hydroxyl group. This reaction is favored by the excellent leaving group (water) on the beta-carbon. Thus PLP catalyzes the removal of water in Serine dehydratase:
The resulting aminoacrylate is then hydrated to give pyruvate as the product. The analogous reaction is catalyzed by Threonine dehydratase to give 2-oxobutyrate.
Other, non PLP catalyzed, direct deaminations include the hydrolysis of the amide nitrogens of glutamine and asparagine, and the deamination of histidine by histidase to give urocanate and ammonium.
L- & D-Amino acid oxidases are flavoproteins which catalyze the direct oxidation of amino acids in what can be considered detoxification reactions. The aa oxidases exhibit broad specificities. These enzymes occur in the peroxisomes of liver and kidney, where the hydrogen peroxide produced can be eliminated by catalase without damaging the cell:
Note here that unlike mixed function oxidases we are going to peroxide instead of water, and oxygen in peroxide has an oxidation state of -1, so only two electrons are required. So this reaction is not energy intensive - no NADH required, no ATP equiv. lost. But a very toxic product, hydrogen peroxide is produced! (Generally done in isolation in organelles such as peroxisomes which contain catalase to destroy peroxide.)
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.
Last modified 2 May 2010