Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:

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Amino Acid Transamination & Deamination, cont.

Pyridoxal-Phosphate, cont.

Pyridoxal-P catalyzes a variety of other reactions involving amino acids involving destabilization of bonds to the alpha-carbon:

diagram showing Pyridoxal-P  catalyzed alpha carbon reactions

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:

Serine dehydratase reaction diagram

The resulting aminoacrylate is then hydrated to give pyruvate as the product. The analogous reaction is catalyzed by Threonine dehydratase to give 2-oxobutyrate.

Non-PLP Amino Acid Deaminations

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:

amino acid oxidase and catalase reactions

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.)

Urea Cycle

Let's quickly look over the Kreb's Urea Cycle (this is his first cycle, the TCA cycle came later). Urea is synthesized from ammonia, carbon dioxide, and aspartate nitrogen using Kreb's Urea Cycle:

Kreb's Urea Cycle structural diagram

Note that two ATP's are required to synthesize carbamyl phosphate, the first to activate bicarbonate (bicarbonate oxygen attacks the terminal phosphate on ATP, displacing ADP; then ammonia attacks the carbonyl carbon, displacing phosphate {breaking a mixed acid anhydride bond}), the second to provide the activated phosphate on the carbamyl-P itself (a "bicarbonate" oxygen attacks the terminal phosphate of ATP displacing ADP to give carbamyl phosphate). 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:

Kreb's Urea Cycle flow diagram

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):

fumerate + H2O right arrow malate; malate +NAD+ right arrow oxaloacetate + NADH + H+

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:

diagram of the transfer of N from an amino acid through glutamate to aspartate

we also have to produce the ammonia from the first amino acid via glutamate using Glutamate DH,

reaction diagram of the production of ammonia from amino acids via transaminase and glutamate dehydrogenase

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.

Urea Cycle Compartmentation

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.

Tissue Distribution of Amino Acid Catabolism

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.):

Metabilic fate of amino acids diagram

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)!

Alanine Cycle

Much of the nitrogen is carried between the tissues and the liver by the Alanine Cycle:

Diagram showing 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.

Discussion of amino acid metabolism, lean meat, Lewis & Clark etc.

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Last modified 1 May 2013