BMB 401 Lecture 33 Transcript Biochemistry 401 Lecture 33Today we’re going to talk about nucleotide, function, structure and synthesis. We’re going to talk about the de novo pathway, and the salvage pathway. So let’s get started.Let's begin by talking about nucleotide function. Nucleotides are precursors for nucleic acid synthesis, DNA and RNA, and this is what we primarily think about what we think about nucleotides, but they’re also used for energy sources. ATP isa prime energy source for biological processes such as molecular motors and active transport of solutes across membranes. GTP is also sometimes used as an energy source. Nucleotides can also activate biomolecules for synthetic reactions, and we’ve seen this previously, for instance UTP activating glucose and CTP activating alcohol head groups, and we've also seen ATP activating many biomolecules. Nucleotides and their derivatives are also important parts of many of many second messenger systems, such as cyclic AMP and cyclic GMP. Finally, ATP is a source of phosphate for kinase reactions, such as the ones that are involved in regulation. We saw this in glycogen synthesis and degradation, and also in glycolysis, and so nucleotides serve many diverse functions.So now let's talk about structure. We’re going to talk about structure of nucleosides, first. Nucleosides are comprised of two things, sugars and nitrogenous bases. Ribonucleosides are comprised of a ribose sugar and a base,and deoxyribonucleosides are comprised of a deoxyribose sugar and a base, andthe difference is shown here. We have adenosine that’s shown in the top figure and deoxyadenosine that’s shown in the bottom figure. The difference between the two is that deoxyadenosine is reduced at the two prime position, and the arrow is pointing to this position. Now the bases can either be purines or pyrimidines, and we’ll see that in just a minute. Nucleotides are nucleosides plus 1, 2 or 3 phosphates. In the top position we seeadenosine a nucleoside, and in the bottom position we see adenosine monophosphate, AMP. This is also known as adenylate. In this slide, we see the nomenclature of bases, nucleosides, and nucleotides. It'simportant that you know the name of the base, the name of the ribonucleoside or deoxyribonucleoside, and the name of the ribonucleotide, or deoxyribonucleotide.1BMB 401 Lecture 33 Transcript And so it's important that you know the difference between adenosine and adenylate, for instance. Nitrogenous bases fit into two major categories. Purines consist of adenine and guanine, and if you’ll notice, these are structures made up of two rings. Pyrimidines have one ring, and there are three of them, Cytosine, uracil and thymine. To remember this, here are some mnemonics, pure as gold for purines A and G, cut pie, C, U, T are all pyrimidines. I hope this helps.So now let’s look at nucleotide synthesis. There are two major pathways. The firstis the salvage pathway, in which we use recycled bases and attach them to an activated ribose platform, PRPP, which is 5-phosphoribosyl-1-pyrophosphate. The other way is the de novo pathway and this is making bases from scratch. We start with an activated ribose again, PRPP, and we add amino acids, ATP, carbondioxide, and other things to make the finished nucleotide. So in the salvage pathway, 5-phosphoribosyl-1-pyrophosphate plus guanine for instance, yields guanosine monophosphate and pyrophosphate, and so this is driven forward by the pyrophosphate release and subsequent hydrolysis to two molecules of inorganic phosphate. The de novo pathway is little more complex, and pyrimidines and purines use twodifferent strategies. Pyrimidine rings are simple, they’re formed first, and they’re brought to the activated ribose platform PRPP, and the subsequent progression, involving several steps, is first to make UTP and then to make CTP. These are both ribonucleotides that can be used to make RNA. Then UTP is used to make TMP and CTP is used to make dCTP. These are both deoxyribonucleotides. And so what we do first is we make the base first, we attach it to PRPP and then we make ribonucleotides. We use the ribonucleotides as substrates to make deoxyribonucleotides. Now purines are much more complex. They involve two rings. These are made directly on the PRPP platform, first to make the intermediate IMP, then to make ATP and GTP. So, we’re making ribonucleotides first and then to dATP and dGTP to make deoxyribonucleotides. Purine rings are formed piece by piece right on the activated ribose sugar. We're going to go over the primitive de novo pathway first as an overview and then we’re going to go through it bit by bit. We're going to do this with the pyrimidine de novo pathway, but were not going to do the same thing with the 2BMB 401 Lecture 33 Transcript purine de novo pathway. So let's get started with this. First of all we’re going to synthesize an intermediate called orotate. This is comprised of carbamoyl phosphate and aspartate. We’re then going to form orotidylate. This occurs by attaching orotate to PRPP. Then were going to form UMP, uridylate. This happensthrough a decarboxylation of orotidylate and then we’re going to phosphorylate UMP to make UTP, and then were going to aminate UTP to make CTP and these are all ribonucleotides. The next that’s going to happen, we're going to make TPPand dCTP. We're going to start with orotate synthesis, So the first thing we’re going to do is we’re going to make orotate, and it looks likethis. The green portions come from aspartate, and the blue and black portions come from carbamoyl phosphate. Now that's a name that should seem familiar toyou. This is the synthesis of carbamoyl phosphate. The process is essentially the same as what we saw when we looked at the urea cycle. Carbamoyl phosphate synthetase II actually catalyzes the rate-limiting step in pyrimidine de novo synthesis, and so it's really important that you understand this process. This is the cytosolic enzyme that synthesizes carbamoyl phosphate. Do you remember where the other carbamoyl phosphate synthetase was? The one that's involved inurea synthesis is actually in the mitochondrion. This one, that's involved in nucleotide synthesis, is found in the cytosol. And so, just like before, we're going to start with bicarbonate. We're going to phosphorylate it to make carboxy phosphate. We’re then going to aminate it to form carbamic acid, then another phosphorylation
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