Amino Acids

The Acid-Base Chemistry of the Amino Acids

The Amino Acids

Proteins are formed by polymerizing monomers that are known as amino acids because they contain an amine (-NH2) and a carboxylic acid (-CO2H) functional group. With the exception of the amino acid proline, which is a secondary amine, the amino acids used to synthesize proteins are primary amines with the following generic formula.
An amino acid

These compounds are known as a-amino acids because the -NH2 group is on the carbon atom next to the -CO2H group, the so-called carbon atom of the carboxylic acid.

Zwitterions

The chemistry of amino acids is complicated by the fact that the -NH2 group is a base and the -CO2H group is an acid. In aqueous solution, an H+ ion is therefore transferred from one end of the molecule to the other to form a zwitterion (from the German meaning mongrel ion, or hybrid ion).

Zwitterions are simultaneously electrically charged and electrically neutral. They contain positive and negative charges, but the net charge on the molecule is zero.

The Amino Acids Used to Synthesize Proteins

More than 300 amino acids are listed in the Practical Handbook of Biochemistry and Molecular Biology, but only the twenty amino acids in the table below are used to synthesize proteins. Most of these amino acids differ only in the nature of the R substituent. The standard amino acids are therefore classified on the basis of these R groups. Amino acids with nonpolar substituents are said to be hydrophobic (water-hating). Amino acids with polar R groups that form hydrogen bonds to water are classified as hydrophilic (water-loving). The remaining amino acids have substituents that carry either negative or positive charges in aqueous solution at neutral pH and are therefore strongly hydrophilic.

The 20 Standard Amino Acids
NAME STRUCTURE
(AT NEUTRAL pH)
Nonpolar (Hydrophobic) R Groups
Glycine (Gly)
Alanine (Ala)
Valine (Val)
Leucine (Leu)
Isoleucine (Ile)
Proline (Pro)
Methionine (Met)
Phenylalanine (Phe)
Tryptophan (Trp)
Polar (Hydrophilic) R Groups
Serine
(ser)
Threonine
(Thr)
Tyrosine
(Tyr)
Cysteine
(Cys)
Asparagine
(Asn)
Glutamine
(Gln)
Negatively Charged R Groups
Aspartic acid (Asp)
Glutamic acid
(Glu)
Positively Charged R Groups
Lysine
(Lys)
Arginine
(Arg)
Histidine
(His)


Practice Problem 1:

Use the structures of the following amino acids in the table of standard amino acids to classify these compounds as either nonpolar/hydrophobic, polar/hydrophilic, negatively charged/hydrophilic, or positively charged/hydrophilic.

(a) Valine: R = -CH(CH3)2

(b) Serine: R = -CH2OH

(c) Aspartic acid: R = -CH2CO2-

(d) Lysine: R = -(CH2)4NH3+

Click here to check your answer to Practice Problem 1



Amino Acids as Stereoisomers

With the exception of glycine, the common amino acids all contain at least one chiral carbon atom. These amino acids therefore exist as pairs of stereoisomers. The structures of the D and L isomers of alanine are shown in the figure below. Although D amino acids can be found in nature, only the L isomers are used to form proteins. The D isomers are most often found attached to the cell walls of bacteria and in antibiotics that attack bacteria. The presence of these D isomers protects the bacteria from enzymes the host organism uses to protect itself from bacterial infection by hydrolyzing the proteins in the bacterial cell wall.

D-Alanine L-Alanine

A few biologically important derivatives of the standard amino acids are shown in the figure below. Anyone who has used an "anti-histamine" to alleviate the symptoms of exposure to an allergen can appreciate the role that histamine-- a decarboxylated derivative of histidine -- plays in mediating the body's response to allergic reactions. L-DOPA, which is a derivative of tyrosine, has been used to treat Parkinson's disease. This compound received notoriety a few years ago in the film Awakening, which documented it's use as a treatment for other neurological disorders. Thyroxine, which is an iodinated ether of tyrosine, is a hormone that acts on the thyroid gland to stimulate the rate of metabolism.
Histamine L-DOPA

Thyroxine



The Acid-Base Chemistry of the Amino Acids

Acetic acid and ammonia often play an important role in the discussion of the chemistry of acids and bases. One of these compounds is a weak acid; the other is a weak base.

Thus, it is not surprising that an H+ ion is transferred from one end of the molecule to the other when an amino acid dissolves in water.

The zwitterion is the dominant species in aqueous solutions at physiological pH (pH 7). The zwitterion can undergo acid-base reactions, howeer, if we add either a strong acid or a strong base to the solution.

Imagine what would happen if we add a strong acid to a neutral solution of an amino acid in water. In the presence of a strong acid, the -CO2- end of this molecule picks up an H+ ion to form a molecule with a net positive charge.

In the presence of a strong base, the -NH3+ end of the molecule loses an H+ ion to form a molecule with a net negative charge.

The figure below shows what happens to the pH of an acidic solution of glycine when this amino acid is titrated with a strong base, such as NaOH.

In order to understand this titration curve, let's start with the equation that describes the acid-dissociation equilibrium constant expression for an acid, HA.

Let's now rearrange the Ka expression,

take the log to the base 10 of both sides of this equation,

and then multiply both sides of the equation by -1.

By definition, the term on the left side of this equation is the pH of the solution and the first term on the right side is the pKa of the acid.

The negative sign on this right side of this equation is often viewed as "inconvenient." The derivation therefore continues by taking advantage of the following feature of logarithmic mathematics

to give the following form of this equation.

This equation is known as the Henderson-Hasselbach equation, and it can be used to calculate the pH of the solution at any point in the titration curve.

The following occurs as we go from left to right across this titration curve.

* The pH initially increases as we add base to the solution because the base deprotonates some of the positively charged H3N+CH2CO2H ions that were present in the strongly acidic solution.
* The pH then levels off because we form a buffer solution in which we have reasonable concentrations of both an acid, H3N+CH2CO2H, and its conjugate base, H3N+CH2CO2-.
* When virtually all of the H3N+CH2CO2H molecules have been deprotonated, we no longer have a buffer solution and the pH rises rapidly when more NaOH is added to the solution.
* The pH then levels off as some of the neutral H3N+CH2CO2- molecules lose protons to form negatively charged H2NCH2CO2- ions. When these ions are formed, we once again get a buffer solution in which the pH remains relatively constant until essentially all of the H3N+CH2CO2H molecules have been converted into H2NCH2CO2- ions.
* At this point, the pH rises rapidly until it reaches the value observed for a strong base.

The pH titration curve tells us the volume of base required to titrate the positively charged H3N+CH2CO2H molecule to the H3N+CH2CO2- zwitterion. If we only add half as much base, only half of the positive ions would be titrated to zwitterions. In other words, the concentration of the H3N+CH2CO2H and H3N+CH2CO2- ions would be the same. Or, using the symbolism in the Henderson-Hasselbach equation:

[HA] = [A-]

Because the concentrations of these ions is the same, the logarithm of the ratio of their concentrations is zero.

Thus, at this particular point in the titration curve, the Henderson-Hasselbach equation gives the following equality.

pH = pKa

We can therefore determine the pKa of an acid by measuring the pH of a solution in which the acid has been half-titrated.

Because there are two titratable groups in glycine, we get two points at which the amino acid is half-titrated. The first occurs when half of the positive H3N+CH2CO2H molecules have been converted to neutral H3N+CH2CO2- ions. The second occurs when half of the H3N+CH2CO2- zwitterions have been converted to negatively charged H2NCH2CO2- ions.

The following results are obtained when this technique is applied to glycine.

Let's compare these values with the pKa's of acetic acid and the ammonium ion.
CH3CO2H pKa = 4.74
NH4+ pKa = 9.24

The acid/base properties of the a-amino group in an amino acid are very similar to the properties of ammonia and the ammonium ion. The a-amine, however, has a significant effect on the acidity of the carboxylic acid. The -amine increases the value of Ka for the carboxylic acid by a factor of about 100.

The inductive effect of the a-amine can only be felt at the a-CO2H group. If we look at the chemistry of glutamic acid, for example, the a-CO2H group on the R substituent has an acidity that is close to that of acetic acid.

When we titrate an amino acid from the low end of the pH scale (pH 1) to the high end (pH 13), we start with an ion that has a net positive charge and end up with an ion that has a net negative charge.

Somewhere between these extremes, we have to find a situation in which the vast majority of the amino acids are present as the zwitterion -- with no net electric charge. This point is called the isoelectric point (pI) of the amino acid.

For simple amino acids, in which the R group doesn't contain any titratable groups, the isoelectric point can be calculated by averaging the pKa values for the a-carboxylic acid and a-amino groups. Glycine, for example, has a pI of about 6.

pI = 2.35 + 9.78 = 6.1
2

At pH 6, more than 99.98% of the glycine molecules in this solution are present as the neutral H3N+CH2CO2H zwitterion.

When calculating the pI of an amino acid that has a titratable group on the R side chain, it is useful to start by writing the structure of the amino acid at physiological pH (pH 7). Lysine, for example, could be represented by the following diagram.

At physiological pH, lysine has a net positive charge. Thus, we have to increase the pH of the solution to remove positive charge in order to reach the isoelectric point. The pI for lysine is simply the average of the pKa's of the two -NH3+ groups.

pI = 9.18 + 10.79 10.0
2

At this pH, all of the carboxylic acid groups are present as -CO2- ions and the total population of the -NH3+ groups is equal to one. Thus, the net charge on the molecule at this pH is zero.

If we apply the same technique to the pKa data for glutamic acid, given above, we get a pI of about 3.1. The three amino acids in this section therefore have very different pI values.
Glutamic acid (R = -CH2CH2CO2-): pI = 3.1
Glycine (R = -H): pI = 6.1
Lysine (R = -CH2CH2CH2CH2NH3+): pI = 10.0

Thus, it isn't surprising that a common technique for separating amino acids (or the proteins they form) involves placing a mixture in the center of a gel and then applying a strong voltage across this gel. This technique, which is known as gel electrophoresis, is based on the fact that amino acids or proteins that carry a net positive charge at the pH at which the separation is done will move toward the negative electrode, whereas those with a net negative charge will move toward the positive electrode.

The Last One

by Austin Repath

The old man hunched forward, his head slightly bent to one side. He wondered where he was. Maybe this was all a dream; maybe he was dead. For years he had lived alone in a dark cave near an ancient swamp, and now.

He looked about the great hall. Although the hall was lit only by candlelight, there were no shadows; everything seemed filled with its own translucent light. The old man narrowed his eyes and studied the people about him. They too seemed to glow with some inner light.

One of them smiled at him. He frowned back and scratched his beard. For some reason he couldn't understand he was the guest of honor. They'd told him that he was the last one, whatever that meant. He had refused to talk to any of them, afraid they might find out what he had done far back in that other time.

Suddenly a great hush fell over the hall, and in through the marble archway walked a white haired woman in flowing white robes. Slowly she took her place at the far end of the hall on a great throne-like chair similar to the one at the other end of the hall on which the old man was seated.

"To all of you, welcome." The voice of the white lady rang like crystal throughout the hall. Then she looked toward the old man. "Long have we searched for you, and now at last you are found. You are most welcome.

The old man stared back at her. She was old beyond imagining, yet in the soft candlelight she looked young and beautiful beyond belief. He could not remember ever having seen her before, yet she awoke deep within him, long forgotten memories.

"This is a day of celebration," continued the white lady, "and as is our custom, we begin our celebration with the story of that other time." She paused for a moment, then began, "Far back in that other time, humankind was like a butterfly about to emerge from its dark cocoon. But it was a timid butterfly, afraid to leave the comfort of what it knew. Back in that other time humankind had everything they needed to go forward, yet they clung to their old ways. In fear they held to their old beliefs.

"It is hard for you who live in love to imagine the iron grip of fear, but humankind lived in fear, and it was this that held them back. Humankind could no longer love because fear filled their hearts. And so that this might be taken from the hearts of men, they were taken into that which they feared the most - world's end. World's end did not come from war as everyone had expected. It came about in a way that put an end to war altogether. In that other time, poisoned by men's fears, the oceans began to die.

The old man leaned forward. He knew that other time. It was his time - and it hadn't been fear that had killed the oceans. "I was there when the oceans were dying," he wanted to shout at them. "When scientists discovered that industrial waste draining into the oceans was killing everything in them even the almost invisible plankton. I was there when they discovered something that stunned the world the fact that most of our oxygen came not from the trees, but from these insignificant sea creatures called plankton, and that once they died, except for a few animals that lived deep in the forests, everything that breathed oxygen would die as well. I was there when the scientists predicted we had only two more years to live."

The old man wanted to stand up and cry out what he knew, but he didn't. He was afraid to. He was afraid they'd discovered what he had done.

The white lady continued, "It was fear that brought the nations of the world together. In that other time nothing else but fear could have united the world. For what was the point of fighting if in two years they'd all be dead? So the nations of the world met together and after many days and nights of talking and arguing, they selected four leaders to coordinate the task of saving the oceans.

"First the four leaders brought together experts from every country to work together on the problem. Then they ordered that food be shipped to wherever people were hungry. No one needed to feel the fear of hunger. But the most important thing the four did was to see that everyone on earth knew what was happening. This was the first step in breaking the fear that gripped the world - that everyone know the truth. They did this with a device that allowed people to see and hear each other over great distances."

The old man smiled to himself at the white lady's description of a television set. He remembered how it had been. Every family on earth had been issued one, and each day at the appointed hour, they'd watched to see if an antidote for the dying oceans had been discovered.

Finally, with only 14 months left, the leaders announced that a chemical had been found that could perhaps neutralize the poison. The old man remembered that well. Day and night they had worked to make enough of the chemical to spread across the oceans. Everyone had worked hard and yet they seemed to have enjoyed it. During that time even strangers would stop and talk to one another.

Then had come the fateful day when thousands and thousands of ships loaded with the chemical headed out to sea. After that there were weeks of waiting to see if it had worked. The old man had turned 22 on the day that the results were announced. The oceans had not been neutralized. They had failed!

Twenty-two years old, his life just beginning, and now it was over. The old man clenched his fist at the memory of that other time. He looked at the white lady as if it was her fault.

But she was continuing with her story. "The nations of the world did fail to save the oceans, but it wasn't really a failure; it only seemed that way. You see, the nations of the world had stopped fighting. War had come to an end. That was the real success!"

The old man stared at her. He had been there and they had failed; they had all been faced with death. He listened to her in disbelief as she went on.

"The people of the world were shocked, and angry. They had tried so hard, and they were still under the illusion that they had failed. The truth was that for the first time in the history of the world, the peoples of earth had worked together with one purpose. They now trusted one another. They were ready for the next step.

The old man looked at her darkly. He remembered when the four leaders had announced the earth had only five months of oxygen left. They had talked about the possibility of something unexpected happening. Maybe even that humans might evolve beyond the need for oxygen. That had been utter absolute nonsense, the old man knew. He glared at the white lady as if daring her to tell him differently.

The white lady smiled toward him, and for some reason he suddenly remembered that one of the leaders had been a woman - a woman who had won world acclaim for her cure for cancer. A white haired lady whom one reporter had dubbed the fairy godmother of the world - and ever after that she had been known affectionately as simply, the godmother. Being the senior member, she had been the last of the four to speak. The words she had once spoken came into his head:

"There is a way out. We can change. I know most of you believe it is impossible to really change, to become something different than you are, but it's not.

"But First we have to let go of our old beliefs. We must accept the fact that each of us is responsible for what has happened. We cannot blame one another.

The old man didn't want to even think about what he had done, and he let his mind drift away onto other things - but: the white lady drew him back with her words: "Those last few months were the most important times in man's history. People began to understand that it was their own fear that was polluting the world, killing the oceans. Even the righteous began to see that they too acted out of fear rather than love.

"However even in their darkest moment humankind was capable of a magnificent gesture. People from all over the world began sending messages to the godmother. 'Find some way of saving the children. Our children are young and unafraid. They are not tied to old ways and for them nothing is impossible. Perhaps they can change. Maybe deep in the forests there is enough oxygen for them to live at least long enough to try. Find some way of saving the children.' Here was a world-wide sense of caring beyond self and family. I t was the sign that the butterfly was getting ready to leave its cocoon."

The white lady stopped and studied the old man for a long time. He looked frantic as if he wanted to run from the hall. She knew that she had to reach him now or he'd be lost forever. She'd have to risk bringing him into her story.

"And so the children were sent into the forests - whatever large and ancient trees could still be found, for that was where there might be enough oxygen for them to live. And with each group of children was sent a strong young man, a young man who was loving and fearless - a man chosen because he had promised to protect the children."

Inside the old man's head, the words were pounding, STOP! STOP!

"And with us tonight is one of these men who long ago was sent out to protect the children. He is our guest of honor!"

Everybody turned and looked toward the old man. Words were exploding inside his head, "it's a lie. It's a lie." Suddenly he realized he was on his feet, shouting at them all, "It's a lie!" He knew now he would have to tell them what he had done. "Yes, I was one of these men who went to protect the children. But I wasn't fearless or loving. I went because I was afraid. I didn't care about the children, I just didn't want to die. Can you understand that?!

"And I wasn't chosen - I begged, begged them to let me go. Then came that strange day in the forest. Everything filled with a blinding light. It was so powerful it had to have been a light blast from some horrendous holocaust, and I ran. Yes, I left the children, ran and hid in a cave. I've lived in that cave ever since. I don't know what happened to the children.

Suddenly he had a strange thought. These people looking at him were those children grown to maturity. They had come back to judge him.

"Yes," he said, the words coming slow and broken. "I left you to perish in that blinding light. All I thought about was myself. I was terrified with my own fear. That's why I did it. I wish it could have been different." He sank back into his chair and stared blankly at the floor, while tears of shame, guilt and then relief ran down his race. He had never told anyone before.

The white lady waited until the old man raised his eyes, then she asked, "Would you like to know what happened when you ran back into the cave?"

He nodded. "Would you like to know how my story ends?"

"Yes, I would," he said, leaning forward in his chair.

"Well, after the children went into the forest, those of us who remained behind had no choice but to accept our fate. As we did so, our fear disappeared! We were no longer afraid, even of world's end. A great peace swept across the earth.

"Then the last day arrived. Everyone gathered at the appointed hour in front of their sets, and the face of her whom they called the godmother appeared to each of them, and here is what she said. 'Your work is all but done. You have changed war into peace, and transformed fear into trust. To each of you I say well done. The next step is so simple your minds will deny it. You have but to join together, speak with one voice, and open yourselves to the power within you.

"For you now stand ready to know the full truth of your being - you now can be trusted with the power of the universe that has been locked within your hearts since time began. You are now ready for a happier world. Come let us cross over together. Let us open our hearts to each other and speak with one voice!" And these were the words that were spoken by every person on earth. We the people of earth, of one mind and heart open ourselves to the power of love and truth."

"And with these words, spoken at the same moment in time by every loving person on earth, everything was changed. The butterfly left its cocoon of fear and darkness, and the earth shone like a luminous pearl in the heavens." The old man sat there, nodding his head slowly. He now knew what happened on that strange day in the forest. That flash of blinding light had been the bursting force of that power locked within the human heart. But that moment of human destiny had passed him by. In fear he had run back to the cave, while they had gone forward.

He longed for another chance. And as he sat there the godmother got up and began walking towards him, and he knew that it was true - they had come back for him.

He got to his feet and walked forward to meet her, his hands outstretched in greeting. When she took his hands in hers and smiled into his eyes, he felt himself filling with light and his heart bursting with joy. A faint smile trembled on his lips as all about him he could hear singing and laughter. The celebration had begun. The last one had crossed over into the light.