The 10-Minute Urea Cycle This tutorial will show you that the urea cycle, perhaps the most important cycle in nitrogen metabolism, can be mastered in 10 minutes. Follow the instructions and convince yourself by the test at the end that you know this cycle. Another important point of the tutorial is to show you that learning structures are the key to developing a confidence in biochemical pathways. So, start your clock and click 1 for the first slide.

COO CH2 H3N-C-H NH HN=C NH2 – Ornithine H2N NH2 C O Urea O Citrulline As with most biochemical pathways, the urea cycle should be mastered by working backwards. Let’s start by drawing the last component in the pathway …Arginine (click 1). As you study the structure of arginine try to imagine all of the intermediates in the pathway built into this one molecule. Perhaps some color will help. Arginine is the immediate source of urea. Can you see urea in arginine. Oh, excuse me!. Click 1 to see urea. Now, can you see urea in arginine (click 1). The oxygen is obtained from H2O when the urea molecule is hydrolyzed free by the enzyme arginase. COO CH2 H3N-C-H NH HN=C NH2 – + Ornithine H2N NH2 C O After the urea is removed by hydrolysis, what remains is ornithine (click 1). Ornithine reacts with carbamoyl-PO4 to form citrulline (click 1). Now that you know how three compounds fit into the arginine molecule, its time to assemble arginine (click 1 to go on). Urea O Citrulline

+ Pi Ornithine Citrulline O C OPO3= H2N COO- H3N-C-H COO- H3N-C-H CH2 Arginine assembly starts with ornithine (click 1). First, you must make the carbamoyl-PO4 that condenses with ornithine (click 1). As you can see below, carbamoyl-PO4 is assembled from NH4+ and CO2 using 2ATPs as an energy source and the enzyme carbamoyl-PO4 synthetase I . NH4+ + CO2 + 2ATP H2N C O OPO3= + 2ADP + Pi COO- CH2 H3N-C-H NH NH2 + O=C Carbamoyl-PO4 reacts with ornithine to form citrulline (click 1). Phosphate is liberated in the reaction (click 1). Once citrulline is formed , all you have to do to make arginine is to replace the oxygen on the citrulline with a nitrogen group (click 1). COO- CH2 H3N-C-H NH3 + + Pi Ornithine Citrulline

+ COO− CH2 H3N-C-H NH NH2 O=C COO − CH2 H3N-C-H NH NH2 =C COO H-C-N In the second step of the pathway the oxygen on the citrulline is replaced with a NH3 group from aspartate (click 1). A complex must be formed that allows the transfer to occur smoothly (click 1). Forming the complex requires ATP, but no phosphate group is transfered (click 1). COO− CH2 H3N-C-H NH NH2 + O=C COO − CH2 H3N-C-H NH NH2 + =C COO H-C-N COO − CH2 H3N-C-H + ATP AMP + PPi + Now you can see arginine in the product. The molecue that forms is called “argininosuccinate” Where’s the succinate? Click 1 to see.

COO– CH HC COO– CH2 HO-C-H COO– CH2 C=O COO– CH2 H3N-C-H Fumarate We now made urea, but there is still a little work left. Cyclic pathways, as their name implies, return back to the starting compounds. When we split the succinate from the argininosuccinate, a pair of electrons went with the NH3 group and we were left with “oxidized” succinate, better known as “fumarate” (click 1). Sound familiar. It should. This is Krebs cycle stuff and we are in the mitochondria. Our objective is turn fumarate back into aspartate (click 1). To do this we must make OAA (click 1). To make OAA we need L-malate (click 1). Bingo! Now all we need is to coenzymes and cofactors to connect all of these intermediates as you can see it happen. COO– CH HC COO– CH2 HO-C-H COO– CH2 C=O COO– CH2 H3N-C-H + NAD+ NADH Glutamate -Kg H2O Fumarate L-Malate OAA Aspartate Check you watch. You just learned the urea cycle is less than 10 minutes. Click 1 and see how well you can do on a short test.

See what you learned. Click for the answer after reading the question. 1. Name 5 -amino acids that are required in the urea cycle. There are 3 traditional, arginine, aspartate, and glutamate, as well 2 non-traditional: ornithine and citrulline. By traditional is meant amino acids that appear in proteins. 2. What is the function of glutamate in the cycle? Glutamate is needed to regenerate aspartate from OAA. 3. All told, how may ATPs are needed to make one molecule of urea? Three are needed . Two to make carbamoyl-PO4 and one to provide energy for the aspartate condensation with citrulline. 4. Why is the urea cycle referred to as a “bicycle”? There are actually 2 cycles going on. One takes ornithine to arginine and returns arginine to ornithine. The second takes fumarate from the argininosuccinate and returns it to aspartate.