Week Two Lecture Biochemistry 560B Dr. Charles Saladino As we start this second week of biochemical deliberations, let us state the obvious. Amino acids are the building blocks of proteins. It therefore puzzles me as to why the Gropper text discusses protein structure before it talks about amino acids, because the structure of proteins for the most part dependent upon its amino acid sequence. Thats one of several reasons by I chose the Devlin text. With that in mind, let us begin this weeks work with the topic of amino acids. It is a big topic. We will save protein structure for next week. The Repertoire of Amino Acids

There are twenty common amino acids, those for which at least one messenger RNA codon exists in the genetic code. We will see in lecture material presentedtoward the end of the course that transcription and translation of the DNA code results in polymerization of the amino acids into a given linear sequence will result in the formation of a characteristic protein. These common twenty amino acids all have the same general structure shown above. As would be expected, there are four attachments to a central carbon atom. One such group is a carboxyl. (In fact the central carbon is also known as the -carbon, because it is one carbon away from a carboxyl group.) An amine group and a hydrogen atom are also attached to the central carbon. The fourth group (R group) is the only place where different amino acids vary in their structure. In fact, the simplest amino acid, glycine, has only a single hydrogen as its R group. If you look at Figure 3.3 in Devlin, you can observe the structure of the twenty common amino acids. Notice that they are classified by the chemical nature of their respective R groups. For example, notice the carboxyl in

the R group of glutamic acid (Glu) and aspartic acid (Asp), giving both amino acids the property of being acidic. These are potentially polar, acidic, and charged in their R groups, whereas lysine (Lys) is potentially a basic, polar, charged amino acid, containing an amine as part of the R group. We must realize, however, that different texts classify the amino acids differently, (ex. polar and basic, like arginine); but still amino acids are classified solely by their R groups. For the first exam, be able to recognize each amino acid by R group. However, you will not have to draw any amino acids on any exam. You might have noticed that I have been using the term common amino acids for the twenty that are shown in FigureTable 3.3 of Devlin. The reason for the word common is that for a variety of reasons, amino acids can be modified as is necessary for the given roles they must play in their respective proteins. Thus, we shall see hydroxyproline and hydroxylysine in the structure of collagen, as well as the joining of two cysteines at their sulfhydryl groups to form the disulfide bond of the modified amino acid, cystine. We will discuss such amino acids later on. Essential and Non-Essential Amino Acids Although there are gradations to classifying amino acids as essential and non-essential, when we see those terms we can generally attribute them to whether or not they can be synthesized in the body. Thus, essential amino acids generally must be obtained through the diet, because they can not be formed in the human body. Non-essential amino acids can be synthesized and are, therefore, classified as non-essential. Your text on page 1103 discusses the essentiality issue, and you can also refer to Table 27.2. Of interest is the PKU condition (phenylketonuira), wherein the enzyme phenylalanine hydroxylase is not present to covert Phe to Tyr. Retardation would occur without Tyr in the diet. This inborn error of metabolism shows how a normally non-essential amino acid can become essential. Isomers of Amino Acids

What you have in the above diagrams are D and L isomers of the same amino acid. Superimposed on the left and right hands, respectively, are two mirror images, which if you tried to slide them over on top of one another, would not be superimposable. Thus, the two fulfill the criteria for being optical isomers (enantiomers) of each other. This is shown again in the second diagram above. The D and L forms are so named based on how they affect polarized light. There is an important practicality of this as it regards human nutrition. That is, only the L-forms of amino acids are incorporated into human protein. We do not have the enzymes to make use of the D forms in this manner. Why this turned out to be part of the grand design is not understood at all, other than the fact that we dont have the enzymes for their utilization throughout metabolism. However, once there was a so-called evolutionary commitment to it, it has remained as such throughout mammalian species. Amino Acids and pKa

It is extremely important that you understand that under physiological conditions, the -amino group is protonated to its ammonium ion form, whereas the carboxyl group is not protonated, but rather it is in its ionic form. In our first weeks lecture, I explained that the pKa of a chemical group is the pH at which there is 50% dissociation of that group. This becomes biochemically relevant wherever there is an available group that can ionize in the amino acid. This potentially means the -amine group, the -carboxyl group, and any ionizable group in the R side chain, including the amine, carboxyl, as well as the sulfhydryl of cysteine. Now, what we are about to discuss is not just a chemical exercise. It is the basis for understanding what acid groups are available for bonding, when an overall protein structure is being formed. As we go through the course, we will observe over and over again that the proper folding of a protein, especially an enzyme, is critical to its proper function. Thus, whether groups ionize or do not affects their bonding abilities. That in turn affects the protein structure. So let us take a look at how this pKa affects ionization. If we look at the graph above, we notice that the pH at which a system operates affects the degree of ionization. Let me be specific. A carboxyl group, depending upon where it is located in a molecule, usually has a pKa that is low, about 3.9, let us say. That means that at pH 3.9, 50% of those particular carboxyl groups are ionized and 50% are not. As we move toward physiological pH, say 7.34, we are relatively at a more basic pH, even though the absolute pH is close to neutral. Remember our definition of a BronstedLowry base? It is one that pulls off/attracts protons. That would increase the degree of ionization (conversion of COOH to COO-). So as we go above the pKa value, the percent ionization increases for the carboxyl group. Observe that in the above graph. Now let us look at the anime group, probably with a high pKa of about 10, where there is 50% NH3 and 50% NH4+. As we go toward physiological pH, the relatively more acidic pH offers more available protons to associate with the amine group to form more ammonium ion. Therefore, the pH relative to the pKa affects ionization of those groups capable of being ionized. Again, observe the above graph. You will also notice that depending upon the pH and the potentially ionizable groups with their respective pKa values, there is a pH range at which the overall charge of the amino acid could be zero. You also see the term, zwitterion, in the graph. Please look that up, and be familiar with it for the first exam, and please do the same for the term isoelectric point, which is not on the graph. In order to help you fully understand what you have been looking at, I have included one more graph below. These are titrations curves. Is this ancient chemistry history perhaps? Actually these are quite simple. What you are doing is adding more and more base (hydroxyl ion), and observe how the pH changes. You will notice that at the pKa points on the graphs for the different ionizable groups, the curve is such that the pH varies

relatively little. Therefore, when you are making a buffer, you want one with a pKa of the weak acid that is close to pH you wish to maintain. When we study hemoglobin and the bicarbonate buffer system of the plasma, you will see more of the practicality of understanding this material.

Also, the pKa value(s) of a compound influence many characteristics of the compound, including its reactivity and solubility. Please remember that in biochemistry the pKa values of proteins and amino acid side chains are of major importance for the activity of enzymes and the stability of proteins. Also, this all relates to our body;s buffer system and why the pKa of our bicarbonate buffer system is so important. Can you make the connection? Let me know. Another important point: We must take careful note of the fact that only free amino acids have a potentially ionizable amine, carboxyl, and R group. When the amino acid is bonded on both sides to other amino acids, as in a protein structure, then the only potentially ionizable group in the structure is in the R group. Then the R side chain must have a potentially ionizable group, as for example, isoleucine does not, whereas arginine does. The R group of isoleucine is highly hydrophobic. To close this part of the lecture, we might ask ourselves why this particular set of 20 common amino acids became the building blocks of a protein. Perhaps another instructor would not probe this type of thinking with you, but I promised you a thought process to be sharpened, and this is an example of it. Briefly, these twenty amino acids offer a rather diverse set of structural and chemical properties. As you will see next week, this diversity endows proteins with the great versatility to assume many functional roles. My

other thought is that potential amino acids might have been eliminated by a biochemical evolution, if those amino acids were excessively reactive within themselves. For example, look up the structure of homocysteine and homoserine on the internet, or one of them is in the Devlin text. You will quickly see that both tend to form five membered cyclic forms. I can tell you that this limits their use in proteins because of steric hinderances. Their alternatives, cysteine and serine, do not easily cyclize, because their ring forms are too small. Follow? Let me know if this is not clear. Whew! Are you OK with this? If not, let me know, because, my friends, I am giving you this info in order to set the stage for your understanding what helps determine higher order protein structure. So please bear with me. Amino Acid Metabolism In this section of the lecture, we will assume the protein has already been digested properly, and that we are starting with amino acids. Protein digestion will be covered briefly and separately next week, but of it is (in my opinion) more for a physiology course rather than for biochemistry. Now if you look briefly at pages 752-797 in Devlin (no you dont have to learn this, just appreciate the complexity), it will appear that amino acid metabolism is very complicated, and in fact it is. I consider it in many ways more complex than either carbohydrate or lipid metabolism. So to keep it within the scope of a reasonable level for this introductory course, I will try to keep our explanation confined to amino acids over all as possible, rather than specific ones. The amino acid pool from which the body has to draw for the synthesis of a variety of biomolecules is relatively small, about 100 g per day. One would at first glance think that the amino acids being recovered from normal protein turnover would be sufficient to replenish these otherwise recyclable compounds. However, that is simplistic and incorrect. In fact, the reason why there is an RDA of about 56 g of protein per day for a 70 kg man, for example, is because many non-protein compounds are synthesized from amino acids, and those compounds are being continuously being depleted. For example, the brain neurotransmitter, serotonin, is synthesized from the amino acid tryptophan. This same amino acid is a precursor for melatonin synthesis in the pineal gland. (You probably remember that this hormone is important in regulating circadian rhythms and sleep.) The purines and pyrimidines of nucleic acids are synthesized from different skeletal components of several different amino acids, including glycine, glutamine, and aspartic acid. There are many more such examples. Thus, we are left with the fact that it is necessary to replace amino acids on a daily basis, because those recovered from the hydrolysis of protein are often used to synthesize non-protein substances in the human body. With this brief introductory thought in mind, let us focus on the critical problem of maintaining an optimal nitrogen balance in the body. Nitrogen balance can be defined as the difference between the amount of nitrogen consumed per day and the amount of nitrogen excreted per day (in grams). This means that because amino acids can not be

stored in great abundance (as can fats, for example), as seen with the relatively small amino acid pool, excess amino acids must be disposed of in such a manner as to ensure that ammonia (derived from amino acid amine group) does not reach toxic levels in the blood plasma. (In the normal adult, plasma levels exceeding 10-80 mcg/dL or 6-47 micromoles/L) are neurotoxic. Here, excess amino acids (or excess protein intake or breakdown) would produce a positive nitrogen balance. (This is in contrast to insufficient protein intake, which would result in a negative nitrogen balance and the degradation of protein to supply the bodys need for amino acids.) The degradation of amino acids occurs basically in a two step process transamination and oxidative deamination, with those amino acids having originated from the digestion of dietary proteins and from the degradation of cellular protein. This set of metabolic reactions takes place in the liver primarily. Transamination Most amino acids are deaminated by transamination, a process whereby their amino group is transferred to an alpha-keto acid to produce a new keto acid plus an amino acid, which is usually glutamate.

Please look at the above diagram, and then look at the one below on the left side of the page. Please relate the two diagrams. This should solidify what I just explained about the overall transamination process. Now look at the lower right diagram, which is the reverse reaction, because transaminations are in fact reversible, if amino acids need to be synthesized and not catabolized. Now I am going to give you sort of an assignment, in that I want you to look at the structure of the amino acid aspartate in the lower right diagram and relate it to the top amino acid in the diagram above. Then look at the oxaloacetate structure and relate it to the top keto acid. The product amino acid will be glutamate again, and the product keto acid will be -ketoglutarate for the above diagram. This is exactly what you would do for the amino acid alanine, except this time you related transamination to aspartate catabolism. Notice in both cases (alanine and aspartate) that the amine group is

being transferred to a keto acid to form glutamate and a new keto acid. This is transamination, and it occurs with almost all the amino acids. However, please note that we have not yet disposed of the amine. It is in the glutamate (the ionic derivative of glutamic acid) where the next step will take place. Before we get to that next step, there is more to note. You need to realize that every amino acid that is transaminated results in the formation of a glutamate (from the -ketoglutarate). As you will see below, glutamate is the only amino acid that can undergo oxidative deamination.

Besides the fact that the above diagram with aspartate shows the reaction in reverse for the formation of amino acids, it also shows the presence of a B6 vitamin derivative, pyridoxyl phosphate. All the transamination enzymes require this as a coenzyme, without which, the enzyme will not function. We have not yet covered enzymes, but we can at least note that each amino acid requires its own transaminase enzyme, all of which require the B6 derivative. The purpose of the pyridoxyl phosphate is to accept the amine group removed from the original amino acid and pass it along to the first keto acid. In other words, the amine is not directly transferred from the original amino acid to the first keto acid. Thus, the role of each transaminase enzyme is to facilitate the transfer of the amino group from the original amino acid, such as aspartate and alanine, as representative examples shown above. For the above two reactions, respectively, the transamination occurs with the transaminase enzyme called alanine aminotransferase

(ALT) and the transaminase enzyme called aspartate amniotransferase (AST). The ALT and the AST might have a familiar ring to them. When a liver profile is being evaluated, plasma ALT and AST values are measured. Clinically, elevation of these enzymes can indicate hepatic damage from a variety of potential sources, for example hepatitis, chronic alcoholism, among many others. Oxidative Deamination This process is in great contrast to the previous transaminations, where an amine group was only transferred from one molecule to another. Now the -amine group is liberated from the amino acid glutamate, resulting in the formation of free ammonia. Glutamate has a special quality, in that it is the only amino acid that undergoes a rapid oxidative deamination, catalyzed by the enzyme, glutamate dehydrogenase. This enzyme can use either NAD+ or NADP+ as its coenzyme (not a usual occurrence to be able to use more than one coenzyme). Like the transaminases, glutamate dehydrogenase is reversible, depending upon: 1) a positive vs. negative nitrogen balance; 2) the availability of the oxidized (ex NAD+) vs. reduced form (NADH) of the coenzyme; 3) the plasma levels of ammonia; 4) the availability of ketoglutarate; 5 GDP and ADP (stimulators of the enzyme) vs. ATP and GTP (inhibitors of the enzyme. Note: this type of regulation will be covered in the enzyme lecture.). So, for example, after a meal containing protein, hepatic glutamate levels rise, and the reaction proceeds in the direction of ammonia formation. Again, less there be no confusion, I have been talking about the amino acid glutamate, the ionized form of glutamic acid. This is always a product of transamination. When free ammonia is released from individual cells in the liver, it is a simple matter to get it to the liver mitochondria, where some of the reactions that form urea take place. Now an important distinction needs to be made. In tissues other than the liver, like the brain and muscle, after transamination and then oxidative deamination of glutamate by the enzyme glutamate dehydrogenase have occurred, another important energy-requiring reaction takes place, which is carried out by the enzyme glutamine synthase. In this reaction, a second ammonium ion is incorporated into an existing glutamate to form the amino acid glutamine. (Remember, glutamate and glutamine are two different amino acids. Review their structures to help you see the difference.) Not surprisingly, glutamine can account for a full fifty percent of the circulating amino acids, because ammonia is carried in the form of glutamine from various tissues like the brain and muscle to the liver for urea formation. Yes, I am saying that glutamine is the main means of ammonia transport in the blood (besides low levels of circulating ammonia), in so far as ammonia (in the form of an amine) is destined for urea formation. You can imagine that the formation of glutamine from glutamate in the brain is a most critical mechanism for ammonia removal in the brain, again because ammonia is neurotixic. (You might explore this in the Discussion Board, if you wish, perhaps with some references.) Anyway, once the glutamine reaches the liver, the amine group is removed from the glutamine by a glutaminase enzyme, and that free ammonia can be

incorporated into the urea cycle. There are tissue-specific forms of this enzyme, called isoenzymes, that concept to be discussed during the enzyme lecture. Thus, the kidney also contains its own glutaminase whose action is the same as the liver glutaminase. Urea Cycle Although some of the ammonium ion formed in amino acid breakdown is consumed in the synthesis of nitrogen compounds, the excess in most terrestrial vertebrates is converted into urea within the liver mitochondrial matrix, released into the blood, sent to the kidney, and then excreted. That makes us urotelic. This urea synthesis occurs in the urea cycle, the steps of which I do not expect you to memorize. However, please learn the structure of urea, as shown below.

The structure of urea is simple, a carbonyl flanked by two amine groups. Without going into the details of the cycle, the carbonyl group is derived from bicarbonate ion (HCO3-), whereas one nitrogen atom comes from aspartate degradation. The second nitrogen atom is derived directly from the free ammonium ion that was liberated during oxidative deamination of the various amino acids, as well as the glutaminase action upon the side chain amide group of glutamine (do you remember what an amide is?) or from other aminoa cids. As eluded to above, part of the urea cycle takes place in the mitochondria. Specifically, the first two steps of the cycle take place there, whereas the remaining enzymes for that cycle are located in the cytosol. Again, urea formation in the liver is quantitatively the most important disposal route for ammonia. Once urea is formed, it is transported to the kidneys, where it enters the glomerular filtrate for excretion. Please look up the urea cycle in your text, not to memorize, but to appreciate its complexity and get an idea how urea formation occurs. Metabolic Sources of Ammonia So where does all this problem ammonia find its origin in the body? The answer is several sources: 1) A big source is the transamination/demination sequence in the catabolism of amino acids. 2) Bacterial degradation of urea occurs in the intestine, releasing ammonia, which is absorbed and sent via the portal vein to the liver. Most of that ammonia is removed by the urea cycle. 3) The side chain amide of glutamine (again, review its structure) provides the ammonia, as discussed in detail above. In the kidney, it is almost immediately excreted as such. Further, by the NH3 picking up a proton to form NH4+, an important aspect of the bodys acid/base balance is achieved.

Finally, a certain amount of glutamine either from the blood or from dietary protein is acted upon by an intestinal glutaminase, releasing ammonia, which finds its way to the liver, again via the portal system. 4) During the catabolism of purines and pyrimidines of the nucleic acids, amines are released from the ring strcutures as ammonia. 5) Finally, ammonia can be derived from amines obtained from the diet and from hormones and neurotransmitters, such as the serotonin and melatonin we mentioned earlier. Amino Acid Transport The final subject of this lecture is the transport of amino acids into the gut. However, this I will assign as a reading. Please refer to pages 1046-1048 for an explanation of this system. However, you do not have to memorize able 25.8. Just get a feel for the fact that there are many transport systems. Some (but not all) of the important take home points regarding amino acid transport can be summarized as: 1) Most of the absorption of amino acids is in the proximal small intestine. 2) An energy-requiring protein carrier molecule is one requirement for amino acid transport. There is some specificity of the carrier for a given amino acid, but that specificity can be overlapping for more than one amino acid. 3) The carrier also requires a Na+/K+ cotransporting system. Please read this carefully at the bottom of page 1047. 4) Consider the different side chains as grouping the amino acids for their transport. 5) Consider that there is a varied affinity of the various amino acids for the different transporters, not discussed in the text. You will definitely see this concept again when we discuss enzymes. Final Thought You might have noticed, despite the volume and detail of this lecture, that I have not included a discussion of amino acids as precursors to other biomolecules, except but briefly. However, when we study gluconeogenesis and protein synthesis, then this subject will be addressed, at least in part. Again, but looking throught pages 752797, you will quickly understand why I believe that more details about the metabolism of amino acids is beyond the scope of an introductory course in biochemistry.