Amino Acids: Structure and Function Case Based Learning #1 Required Reading: Some resources and a limited reference

list for this case are provided below in order to assist you during the process of problem solving. This reference list is not exhaustive, and additional literature search will be productive and is strongly encouraged. You are also encouraged to consult with the resource faculty for assistance in filling any gaps in your understanding of the material. We must emphasize, however, that the responsibility for, and benefits from, learning and .mastering the necessary knowledge and content relevant to this case are yours 1. Workgroup Notes (see below) :From: The Medical Biochemistry Page 2. Lieberman and Marks Text: Chapter 6, pps 73-82, Chapter 7, pps. 92-102 3. Emedicine article on alpha-1-antitrypsin deficiency, selected sections (see below). Case Ingrid Ernster, a 44-year-old white female, complains of shortness of breath, which she has experienced for the past year. She tires easily, even after modest exertion. Ms. Ernster tells the doctor that she smoked a pack a day since she was 16., however discontinuing smoking did not change the shortness of breath or fatigability. She denies any chronic productive cough or sputum production Pulmonary function tests show chronic obstructive lung disease (COPD) and the doctor refers her tests. Serum 1-antitrypsin level was 23 mg/dl (normal 93-224 mg/dl). Electrophoretic testing of serum proteins reveals the presence of Z protein and no M protein. Ingrid Ernster is diagnosed with emphysema due to an 1-antitrypsin deficiency, which is compounded by her long history of smoking. In order to determine the specific mutation in the 1-antitrypsin gene, a DNA analysis was done: Polymerase Chain Reaction (PCR) and restriction mapping test reveal a substitution of A for G in exon 5 of the gene encoding 1-AT. This mutation replaces a glutamic acid at position 342 in the 1-antitrypsin protein with a lysine. This is consistent with the ZZ phenotype.

Case Objectives 1) Understand the general chemical properties of amino acids as building blocks of proteins in humans 2) Discuss the different classes of amino acids and how the different side chains give them distinct properties 3) Define primary, secondary, tertiary and quaternary structure of proteins 4) Explain the difference between a conservative and a nonconservative amino acid substitution, and the consequences of such mutations on the structure and function of proteins. 5) Understand the pathophysiology of alpha-1-antitrypsin deficiency. Core Concepts 1. Covalent bonds 2. Hydrogen bonds 3. Van der Waals bonds 4. Water is a polar molecule 5. Water solvates polar molecules 6. 20 amino acids build proteins. Amino acids in solution at neutral pH are predominantly dipolar ions; the amino group is protonated and the carboxyl group is dissociated. 7. Amino acids vary in size, shape, charge, hydrogen-bonding capacity, and chemical reactivity. 8. Amino acid having aliphatic side chain: gly, ala, val, leu, ile, pro. 9. Amino acids having aromatic side chains: phe, tyr, trp. 10. Amino acids with sulfur containing side chain: cys, met. 11. Amino acids having aliphatic hydroxyl side chain: ser, thr. 12. Amino acids having basic side chains: lys, arg, his. 13. Amino acids having acidic side chains and their amide derivative: asp, glu, asn, gln. 14. Amino acids are linked by peptide bond to form polypeptide chains. 15. Proteins primary structure; proteins have unique amino acid sequences that are specified by genes. 16. Secondary structure: polypeptide chains can fold into regular structures such as -helix, pleated sheet, turn and loop. 17. The alpha helix is a coiled structure stabilized by inter-chain hydrogen bonds. 18. Beta sheets are stabilized by hydrogen bonding between polypeptide strands. 19. Polypeptide chains can change direction by making loops and turns. 20. Tertiary structure: water-soluble proteins fold into compact structures with polar core.

21. Quaternary structure: polypeptide chains can assemble into multi subunits structures. 22. The amino acid sequence of a protein determines its three dimensional structure. 23. Amino acids side chains have different properties for forming secondary, tertiary, and quaternary structures. 24. Pathological conditions can result from inappropriate conformation: Prion diseases, Alzheimers etc. 25. Proteins fold by stabilization of intermediates. 26. Molecular chaperones are proteins that bind to unfolded and partially folded polypeptides to prevent the improper associations of exposed segments that might lead to non-native folding, aggregation and precipitation in vivo. 27. The proteome is the set of proteins, their isoforms, modifications, and interactions, in any given cell at any given moment. 28. Proteomics is the systemic analysis of cellular proteins. It encompasses the type, functions and interactions of proteins. 29. Cellular functions are performed by structured ensembles of proteins.

Workgroup Notes Chemical Nature of the Amino Acids All peptides and polypeptides are polymers of -amino acids. There are 20 amino acids that are relevant to the make-up of mammalian proteins (see below). Several other amino acids are found in the body free or in combined states (i.e. not associated with peptides or proteins). These non-protein associated amino acids perform specialized functions. Several of the amino acids found in proteins also serve functions distinct from the formation of peptides and proteins, e.g., tyrosine in the formation of thyroid hormones or glutamate acting as a neurotransmitter. The -amino acids in peptides and proteins (excluding proline) consist of a carboxylic acid (COOH) and an amino (NH2) functional group attached to the same tetrahedral carbon atom. This carbon is the -carbon. Distinct R-groups, that distinguish one amino acid from another, also are attached to the alphacarbon (except in the case of glycine where the R-group is hydrogen). The fourth substitution on the tetrahedral -carbon of amino acids is hydrogen.

Table of -Amino Acids Found in Proteins Amino Acid Symb ol pK1 pK2 pK R (COO (NH2 Grou H) ) p

Structure* Amino Acids with Aliphatic R-Groups

Glycine

Gly G Ala A

2.4

9.8

Alanine

2.4

9.9

Valine

Val V

2.2

9.7

Leucine

Leu L

2.3

9.7

Isoleucine

Ile I

2.3

9.8

Non-Aromatic Amino Acids with Hydroxyl R-Groups Serine Ser S 2.2 9.2 13

Threonine

Thr T

2.1

9.1

13

Amino Acids with Sulfur-Containing R-Groups Cysteine Cys C 1.9 10.8 8.3

Methionine

Met M Acidic Amino Acids and their Amides

2.1

9.3

Aspartic Acid

Asp D

2.0

9.9

3.9

Asparagine

Asn N

2.1

8.8

Glutamic Acid

Glu E

2.1

9.5

4.1

Glutamine

Gln Q Basic Amino Acids

2.2

9.1

Arginine

Arg R

1.8

9.0

12.5

Lysine

Lys K

2.2

9.2

10.8

Histidine

His H Amino Acids with Aromatic Rings

1.8

9.2

6.0

Phenylalani Phe ne F

2.2

9.2

Tyrosine

Tyr Y

2.2

9.1

10.1

Tryptophan

Trp W

2.4

9.4

Imino Acids

Proline

Pro P

2.0

10.6

Backbone of the amino acids is red, R-groups are black Amino Acid Classifications

Each of the 20 -amino acids found in proteins can be distinguished by the Rgroup substitution on the -carbon atom. There are two broad classes of amino acids based upon whether the R-group is hydrophobic or hydrophilic. The hydrophobic amino acids tend to repel the aqueous environment and, therefore, reside predominantly in the interior of proteins. This class of amino acids does not ionize nor participate in the formation of H-bonds. The hydrophilic amino acids tend to interact with the aqueous environment, are often involved in the formation of H-bonds and are predominantly found on the exterior surfaces proteins or in the reactive centers of enzymes.

Acid-Base Properties of the Amino Acids The -COOH and -NH2 groups in amino acids are capable of ionizing (as are the acidic and basic R-groups of the amino acids). As a result of their ionizability the following ionic equilibrium reactions may be written: R-COOH <> R-COO + H+ R-NH3+ <> R-NH2 + H+ The equilibrium reactions, as written, demonstrate that amino acids contain at least two weakly acidic groups. However, the carboxyl group is a far stronger acid than the amino group. At physiological pH (around 7.4) the carboxyl group will be unprotonated and the amino group will be protonated. An amino acid with no ionizable R-group would be electrically neutral at this pH. This species is termed a zwitterion. Like typical organic acids, the acidic strength of the carboxyl, amino and ionizable R-groups in amino acids can be defined by the association constant, Ka or more commonly the negative logrithm of Ka, the pKa. The net charge (the algebraic sum of all the charged groups present) of any amino acid, peptide or protein, will depend upon the pH of the surrounding aqueous environment. As the pH of a solution of an amino acid or protein changes so too does the net charge. This phenomenon can be observed during the titration of any amino acid or protein. When the net charge of an amino acid or protein is zero the pH will be equivalent to the isoelectric point: pI.

Functional Significance of Amino Acid R-Groups In solution it is the nature of the amino acid R-groups that dictate structurefunction relationships of peptides and proteins. The hydrophobic amino acids will generally be encountered in the interior of proteins shielded from direct contact with water. Conversely, the hydrophilic amino acids are generally found on the exterior of proteins as well as in the active centers of enzymatically active proteins. Indeed, it is the very nature of certain amino acid R-groups that allow enzyme reactions to occur. The imidazole ring of histidine allows it to act as either a proton donor or acceptor at physiological pH. Hence, it is frequently found in the reactive center of enzymes. Equally important is the ability of histidines in hemoglobin to buffer the H+ ions from carbonic acid ionization in red blood cells. It is this property of hemoglobin that allows it to exchange O2 and CO2 at the tissues or lungs, respectively.

The primary alcohol of serine and threonine as well as the thiol (SH) of cysteine allow these amino acids to act as nucleophiles during enzymatic catalysis. Additionally, the thiol of cysteine is able to form a disulfide bond with other cysteines: Cysteine-SH + HS-Cysteine <> Cysteine-S-S-Cysteine This simple disulfide is identified as cystine. The formation of disulfide bonds between cysteines present within proteins is important to the formation of active structural domains in a large number of proteins. Disulfide bonding between cysteines in different polypeptide chains of oligomeric proteins plays a crucial role in ordering the structure of complex proteins, e.g. the insulin receptor. Optical Properties of the Amino Acids A tetrahedral carbon atom with 4 distinct constituents is said to be chiral. The one amino acid not exhibiting chirality is glycine since its '"R-group" is a hydrogen atom. Chirality describes the handedness of a molecule that is observable by the ability of a molecule to rotate the plane of polarized light either to the right (dextrorotatory) or to the left (levorotatory). All of the amino acids in proteins exhibit the same absolute steric configuration as L-glyceraldehyde. Therefore, they are all L--amino acids. D-amino acids are never found in proteins, although they exist in nature. D-amino acids are often found in polypetide antibiotics. The aromatic R-groups in amino acids absorb ultraviolet light with an absorbance maximum in the range of 280nm. The ability of proteins to absorb ultraviolet light is predominantly due to the presence of the tryptophan which strongly absorbs ultraviolet light.

The Peptide Bond Peptide bond formation is a condensation reaction leading to the polymerization of amino acids into peptides and proteins. Peptides are small consisting of few amino acids. A number of hormones and neurotransmitters are peptides. Additionally, several antibiotics and antitumor agents are peptides. Proteins are polypeptides of greatly divergent length. The simplest peptide, a dipeptide, contains a single peptide bond formed by the condensation of the carboxyl group of one amino acid with the amino group of the second with the concomitant elimination of water. The presence of the carbonyl group in a peptide bond allows electron resonance stabilization to occur such that the peptide bond exhibits rigidity not unlike the typical C=C double bond. The peptide bond is, therefore, said to have partial double-bond character.

Resonance stabilization forms of the peptide bond

Primary Structure of Proteins The primary structure of peptides and proteins refers to the linear number and order of the amino acids present. The convention for the designation of the order of amino acids is that the N-terminal end (i.e. the end bearing the residue with the free -amino group) is to the left (and the number 1 amino acid) and the Cterminal end (i.e. the end with the residue containing a free -carboxyl group) is to the right. Secondary Structure in Proteins The ordered array of amino acids in a protein confer regular conformational forms upon that protein. These conformations constitute the secondary structures of a protein. In general proteins fold into two broad classes of structure termed, globular proteins or fibrous proteins. Globular proteins are compactly folded and coiled, whereas, fibrous proteins are more filamentous or elongated. It is the partial double-bond character of the peptide bond that defines the conformations a polypeptide chain may assume. Within a single protein different regions of the polypeptide chain may assume different conformations determined by the primary sequence of the amino acids. The -Helix The -helix is a common secondary structure encountered in proteins of the globular class. The formation of the -helix is spontaneous and is stabilized by Hbonding between amide nitrogens and carbonyl carbons of peptide bonds spaced four residues apart. This orientation of H-bonding produces a helical coiling of the peptide backbone such that the R-groups lie on the exterior of the helix and perpendicular to its axis.

Typical -Helix Not all amino acids favor the formation of the (-helix due to steric constraints of the R-groups. Amino acids such as A, D, E, I, L and M favor the formation of helices, whereas, G and P favor disruption of the helix. This is particularly true for P since it is a pyrrolidine based imino acid (HN=) whose structure significantly restricts movement about the peptide bond in which it is present, thereby, interfering with extension of the helix. The disruption of the helix is important as it introduces additional folding of the polypeptide backbone to allow the formation of globular proteins. -Sheets Whereas an -helix is composed of a single linear array of helically disposed amino acids, -sheets are composed of 2 or more different regions of stretches of at least 5-10 amino acids. The folding and alignment of stretches of the polypeptide backbone aside one another to form -sheets is stabilized by Hbonding between amide nitrogens and carbonyl carbons. However, the Hbonding residues are present in adjacently opposed stretches of the polypetide backbone as opposed to a linearly contiguous region of the backbone in the -

helix. -sheets are said to be pleated. This is due to positioning of the -carbons of the peptide bond which alternates above and below the plane of the sheet. sheets are either parallel or antiparallel. In parallel sheets adjacent peptide chains proceed in the same direction (i.e. the direction of N-terminal to C-terminal ends is the same), whereas, in antiparallel sheets adjacent chains are aligned in opposite directions. -sheets can be depicted in ball and stick format or as ribbons in certain protein formats.

Ball and Stick Representation of a -Sheet

Ribbon Depiction of -Sheet

Super-Secondary Structure Some proteins contain an ordered organization of secondary structures that form distinct functional domains or structural motifs. Examples include the helix-turnhelix domain of bacterial proteins that regulate transcription and the leucine zipper, helix-loop-helix and zinc finger domains of eukaryotic transcriptional regulators. These domains are termed super-secondary structures.

Tertiary Structure of Proteins Tertiary structure refers to the complete three-dimensional structure of the polypeptide units of a given protein. Included in this description is the spatial relationship of different secondary structures to one another within a polypeptide chain and how these secondary structures themselves fold into the three-dimensional form of the protein. Secondary structures of proteins often constitute distinct domains. Therefore, tertiary structure also describes the relationship of different domains to one another within a protein. The interactions of different domains is governed by several forces: These include hydrogen bonding, hydrophobic interactions, electrostatic interactions and van der Waals forces.

Forces Controlling Protein Structure Hydrogen Bonding: Polypeptides contain numerous proton donors and acceptors both in their backbone and in the R-groups of the amino acids. The environment in which proteins are found also contains the ample H-bond donors and acceptors of the water molecule. H-bonding, therefore, occurs not only within and between polypeptide chains but with the surrounding aqueous medium. Hydrophobic Forces: Proteins are composed of amino acids that contain either hydrophilic or hydrophobic R-groups. It is the nature of the interaction of the different R-groups with the aqueous environment that plays the major role in shaping protein structure. The spontaneous folded state of globular proteins is a reflection of a balance between the opposing energetics of H-bonding between hydrophilic Rgroups and the aqueous environment and the repulsion from the aqueous environment by the hydrophobic R-groups. The hydrophobicity of certain amino acid R-groups tends to drive them away from the exterior of proteins and into the interior. This driving force restricts the available conformations into which a protein may fold. Electrostatic Forces: Electrostatic forces are mainly of three types; charge-charge, charge-dipole and dipole-dipole. Typical charge-charge interactions that favor protein folding are those between oppositely charged R-groups such as K or R and D or E. A substantial component of the energy involved in protein folding is charge-dipole interactions. This refers to the interaction of ionized R-groups of amino acids with the dipole of the water molecule. The slight dipole moment that exist in the polar R-groups of amino acid also influences their interaction with water. It is, therefore, understandable that the majority of the amino acids found on the exterior surfaces of globular proteins contain charged or polar R-groups. van der Waals Forces: There are both attractive and repulsive van der Waals forces that control protein folding. Attractive van der Waals forces involve the interactions among induced dipoles that arise from fluctuations in the charge densities that occur between adjacent uncharged non-bonded atoms. Repulsive van der Waals forces involve the interactions that occur when uncharged non-bonded atoms come very close together but do not induce dipoles. The repulsion is the result of the electronelectron repulsion that occurs as two clouds of electrons begin to overlap.

Although van der Waals forces are extremely weak, relative to other forces governing conformation, it is the huge number of such interactions that occur in large protein molecules that make them significant to the folding of proteins. Quaternary Structure Many proteins contain 2 or more different polypeptide chains that are held in association by the same non-covalent forces that stabilize the tertiary structures of proteins. Proteins with multiple polypetide chains are oligomeric proteins. The structure formed by monomer-monomer interaction in an oligomeric protein is known as quaternary structure. Oligomeric proteins can be composed of multiple identical polypeptide chains or multiple distinct polypeptide chains. Proteins with identical subunits are termed homo-oligomers. Proteins containing several distinct polypeptide chains are termed hetero-oligomers. Hemoglobin, the oxygen carrying protein of the blood, contains two and two subunits arranged with a quaternary structure in the form, 22. Hemoglobin is, therefore, a hetero-oligomeric protein.

Structure of Hemoglobin

Complex Protein Structures Proteins also are found to be covalently conjugated with carbohydrates. These modifications occur following the synthesis (translation) of proteins and are, therefore, termed post-translational modifications. These forms of modification impart specialized functions upon the resultant proteins. Proteins covalently associated with carbohydrates are termed glycoproteins. Glycoproteins are of two classes, N-linked and O-linked, referring to the site of covalent attachment of the sugar moieties. N-linked sugars are attached to the amide nitrogen of the Rgroup of asparagine; O-linked sugars are attached to the hydroxyl groups of either serine or threonine and occasionally to the hydroxyl group of the modified amino acid, hydroxylysine. There are extremely important glycoproteins found on the surface of erythrocytes. It is the variability in the composition of the carbohydrate portions of many glycoproteins and glycolipids of erythrocytes that determines blood group specificities. There are at least 100 blood group determinants, most of which are due to carbohydrate differences. The most common blood groups, A, B, and O, are specified by the activity of specific gene products whose activities are to incorporate distinct sugar groups onto RBC membrane glycoshpingolipids as well as secreted glycoproteins. Structural complexes involving protein associated with lipid via noncovalent interactions are termed lipoproteins. The distinct roles of lipoproteins are described on the linked page. Their major function in the body is to aid in the storage transport of lipid and cholesterol. Clinical Significances This discussion is not intended to be a complete review of all disorders that result from defects in protein structure and function. Visit the Inborn Errors page for a more complete listing of diseases related to abnormal proteins and also click on the links to the specific examples below for more information. The substitution of a hydrophobic amino acid (V) for an acidic amino acid (E) in the -chain of hemoglobin results in sickle cell anemia (HbS). This change of a single amino acid alters the structure of hemoglobin molecules in such a way that the deoxygenated proteins polymerize and precipitate within the erythrocyte, leading to their characteristic sickle shape. Collagens are the most abundant proteins in the body. Alterations in collagen structure arising from abnormal genes or abnormal processing of collagen proteins results in numerous diseases, including Larsen syndrome, scurvy, osteogenesis imperfecta and Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome is actually the name associated with at least ten distinct disorders that are biochemically and clinically distinct yet all manifest structural weakness in connective tissue as a result of defective collagen structure. Osteogenesis imperfecta also encompasses more than one disorder. At least four biochemically and clinically distinguishable maladies have been identified as osteogenesis imperfecta, all of which are characterized by multiple fractures and resultant bone deformities. Marfan syndrome manifests itself as a disorder of the connective tissue and was originally believed to be the result of abnormal collagens. However, recent evidence has shown that Marfan syndrome results from mutations in the extracellular protein, fibrillin, which is an integral constituent of the non-collagenous microfibrils of the extracellular matrix. Several forms of familial hypercholesterolemia are the result of genetic defects in the gene encoding the receptor for low-density lipoprotein (LDL). These defects result in the synthesis of abnormal LDL receptors that are incapable of binding to LDLs, or that bind LDLs but the receptor/LDL complexes are not properly internalized and degraded. The outcome is an elevation in serum cholesterol levels and increased propensity toward the development of atherosclerosis. A number of proteins can contribute to cellular transformation and carcinogenesis when their basic structure is disrupted by mutations in their genes. These genes are termed proto-oncogenes. For some of these proteins, all that is required to convert them to the oncogenic form is a single amino acid substitution. The cellular gene, RAS, is observed to sustain single amino acid substitutions at positions 12 or 61 with high frequency in colon carcinomas. Mutations in RAS are most frequently observed genetic alterations in colon cancer.

Alpha1-Antitrypsin Deficiency Paul Fairman, MD, Director, Pulmonary Hypertension Service, Professor, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Virginia Commonwealth University Rajiv Malhotra, DO, Fellow, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Virginia Commonwealth University Health System Updated: Apr 16, 2009 Introduction Background Alpha1-antitrypsin (AAT) deficiency, first described in 1963, is one of the most common inherited disorders among white persons. Its primary manifestation is early-onset panacinar emphysema. About 1-3% of patients with diagnosed chronic obstructive pulmonary disease (COPD) are predicted to have alpha1antitrypsin deficiency. Slowly progressive dyspnea is the primary symptom, though many patients initially have symptoms of cough, sputum production, or wheezing. Treatment involves smoking cessation, bronchodilation, and physical rehabilitation in a program similar to that designed for patients with smokingrelated COPD. In addition, intravenous (IV) augmentation therapy with alpha1antitrypsin benefits some patients. Alpha1-antitrypsin deficiency is also an unusual cause of hepatic cirrhosis in children and adults. Pathophysiology The genetic defect in alpha1-antitrypsin (AAT) deficiency alters the configuration of the alpha1-antitrypsin molecule and prevents its release from hepatocytes. As a result, serum levels of alpha1-antitrypsin are decreased, leading to low alveolar concentrations, where the alpha1-antitrypsin molecule normally would serve as protection against antiproteases. The resulting protease excess in alveoli destroys alveolar walls and causes emphysema. The accumulation of excess alpha1-antitrypsin in hepatocytes can also lead to destruction of these cells and ultimately, clinical liver disease.

Frequency United States Alpha1-antitrypsin (AAT) deficiency is 1 of the 3 most common lethal genetic diseases among adult white persons, affecting 1 per 3000-5000 individuals. Severe alpha1-antitrypsin deficiency affects an estimated 100,000 individuals, and approximately 25 million people carry of at least 1 deficient gene. However, less than 6% of severely deficient individuals are currently identified. International Alpha1-antitrypsin deficiency has been identified in all populations, but it is most common in individuals of Northern European and Iberian descent. Similar rates are found among white persons worldwide, with an estimated 117 million carriers and 3.4 million affected individuals. Mortality/Morbidity Specific morbidity and mortality rates are unknown. Not all patients with homozygous deficiency develop symptomatic emphysema or cirrhosis; however, among those who develop symptomatic disease, the mortality rate is high. Race Racial groups other than whites are affected less frequently. Sex Women and men are affected in equal numbers. Age The enzyme deficiency is congenital and has a bimodal distribution with respect to symptoms. It can be seen in neonates as a cause of neonatal jaundice and hepatitis. It can present in infants as cholestatic jaundice and in children as hepatic cirrhosis or liver failure. Alpha1-antitrypsin deficiency is also the leading cause of liver transplantation in children. In adults, alpha1-antitrypsin deficiency leads to chronic liver disease in the fifth

decade. As a cause of emphysema, it is seen in nonsmokers in the fifth decade of life and during the fourth decade of life in smokers. Clinical History Symptoms of alpha1-antitrypsin (AAT) deficiency emphysema are limited to the respiratory system. The initial symptoms of alpha1-antitrypsin deficiency include cough, sputum production, and wheezing. Symptoms are initially intermittent, and, if wheezing is the predominant symptom, patients often are told they have asthma. If recurrent episodes of cough are most prominent, patients may be treated with multiple courses of antibiotics and evaluated for sinusitis, postnasal drip, or gastroesophageal reflux. o Dyspnea is the symptom that eventually dominates alpha1

antitrypsin deficiency. o Similar to other forms of emphysema, the dyspnea of alpha1-antitrypsin deficiency is initially evident only with strenuous exertion. Over several years, it eventually limits even mild activities. o Patients with alpha1-antitrypsin deficiency frequently develop dyspnea 20-30 years earlier (at age 30-45 y) than do smokers with emphysema and normal alpha1-antitrypsin levels. Cigarette smoking accelerates the progression of emphysema in patients with alpha1-antitrypsin deficiency. Symptoms develop about 10 years earlier in alpha1-antitrypsindeficient individuals who smoke regularly. By the time dyspnea becomes the dominant manifestation and a diagnosis is established, most patients will have seen several physicians over several years. Efforts to improve the interval between the onset of symptoms and the diagnosis of alpha1-antitrypsin deficiency have been disappointing. Between 1968 and 2003 a significant improvement has not been noted in the average interval (approximately 8 y), although improvement has been shown in the alpha1-antitrypsin deficiency detection in older individuals.[1 ]

Physical No single physical sign confirms a diagnosis of alpha1-antitrypsin deficiency emphysema. Signs characteristic of increased respiratory work, airflow obstruction, and hyperinflation eventually develop but are dependent on the severity of emphysema at the time of diagnosis. Increased respiratory work is evident as tachypnea, scalene and intercostal muscle retraction, and tripod position. Airflow obstruction manifests as pursed-lip breathing, wheezing, and pulsus paradox. Hyperinflation results in barrel chest, increased percussion note, decreased breath sound intensity, and distant heart sounds. Patients with mild emphysema generally have no abnormal findings on physical examination. o Even moderate disease may be evident only when a

complicating acute infection occurs. o Most of the signs generally considered a part of emphysema (from any cause) are signs of moderate-to-severe disease. o Mild-to-moderate disease is easily missed if the physician relies solely on physical findings. Causes Alpha1-antitrypsin deficiency is an uncommon but not rare disease. It is under diagnosed. The responsible genetic defect affects 1 in 3000-5000 individuals, making it 1 of the 3 most common lethal genetic diseases among whites. (The other 2 common fatal genetic defects are cystic fibrosis and Down syndrome.) Fortunately, not every individual with alpha1-antitrypsin deficiency develops clinically significant disease. The major biochemical activity of the alpha1-antitrypsin molecule is inhibition of several neutrophil-derived proteases (eg, trypsin, elastase, proteinase 3, cathepsin G). Therefore, the protein is more accurately termed alpha1-antiprotease. However, most physicians, and virtually all

patients, refer to the disease as alpha1-antitrypsin deficiency, and doctors and patients often refer to those who are affected as "alphas." Hepatocytes synthesize alpha1-antiprotease. o After its release from the liver, alpha1-antiprotease circulates unbound and diffuses into interstitial and alveolar lining fluids. Its principle function in the lung is to inactivate neutrophil elastase, an enzyme that is released during normal phagocytosis of organisms or particulates in the alveolus. o Alpha1-antiprotease constitutes about 95% of all the antiprotease activity in human alveoli, and neutrophil elastase is considered the protease largely responsible for alveolar destruction. In patients with the Z allele, the alpha1-antitrypsin produced has a lysine substituted for glutamate. This results in spontaneous polymerization within the endoplasmic reticulum of the hepatocyte, which leads to decreased serum levels of alpha1antitrypsin and thus a deficiency of peripheral alpha1-antitrypsin. o Additionally, the accumulation of intrahepatic alpha1antitrypsin is thought to result in apoptosis of hepatocytes. This initially can manifest as laboratory abnormalities, but also can progress to hepatitis, followed by fibrosis and cirrhosis.[2 ] In healthy persons, alpha1-antiprotease serves as a protective screen that prevents alveolar wall destruction. The lungs have a large surface area and are continuously exposed to a high burden of airborne pathogens, which results in a cellular immune response. This is characterized by local release of oxidants and proteases. The presence of alpha1-antiprotease serves to keep these proteases in check and protect the lungs from unregulated protease activity. Individuals with the alpha1antitrypsin genetic defect do not release alpha1-antiprotease from the liver, and serum and alveolar levels of the protein are low. Consequently, alveoli lack antiprotease protection. The imbalance of proteasesantiproteases in the alveolus leads to unimpeded neutrophil elastase digestion of elastin and collagen in the alveolar walls and progressive emphysema. Alveolar cell apoptosis may also play an important role in emphysema pathogenesis. Recent evidence suggests that alpha1-

antiprotease may inhibit alveolar cell apoptosis and protect against emphysema in the absence of neutrophilic inflammation.[3 ] Cigarette smoking accelerates the onset of symptomatic disease by approximately 10 years by increasing the number of neutrophils (and neutrophil elastase) in the alveolus and inactivating the remaining small amounts of antiprotease. Other factors that can accelerate the onset or worsen symptoms of disease include infections and exposures to dust and fumes, which can also cause the recruitment of neutrophils to the alveoli. The production of alpha1-antiprotease is controlled by a pair of genes at the protease inhibitor (Pi) locus. The SERPINA1 (formerly known as Pi) gene responsible for encoding alpha1-antitrypsin is located on chromosome 14 and is highly pleomorphic, with more than 100 allelic variants. The variants are classified based on serum levels of alpha1antitrypsin protein. M alleles are the most common and normal variants. Most patients with clinical disease are homozygous SS or ZZ or heterozygous MS, MZ, or SZ. o Nearly 24 variants of the alpha1-antiprotease molecule have been identified, and all are inherited as codominant alleles. The most common (90%) allele is M (PiM), and homozygous individuals (MM) produce normal amounts of alpha1-antiprotease (serum levels of 20-53 mol/L or 150-350 mg/dL). o The most common form of alpha1-antitrypsin deficiency is associated with allele Z, or homozygous PiZ (ZZ). Serum levels of alpha1-antitrypsin in these patients are about 3.4-7 mol/L, 10-15% of normal serum levels. Serum levels greater than 11 mol/L appear to be protective. Emphysema develops in most (but not all) individuals with serum levels less than 9 mol/L. Other genotypes associated with severe alpha1-antitrypsin deficiency include PiSZ, PiZ/Null, and PiNull. The S gene is more frequent among individuals of Spanish or Portuguese descent, whereas the frequency of the Z gene is highest in patients of Northern or Western European descent. o Patients with the PiSZ phenotype have a 20-50% increased likelihood of developing emphysema compared with MM homozygotes. Serum levels of patients with PiSZ alpha1-antitrypsin deficiency are 75-120 mg/dL.

Patients with the null gene for alpha1-antitrypsin will not

produce any alpha1-antitrypsin and are high risk for emphysema (100% by the age of 30 y). None with the null gene develop liver disease because of a lack of production, and thus accumulation, of alpha1-antitrypsin in the hepatocytes. The null gene is the least common of the known alleles associated with alpha1-antitrypsin deficiency. o Carriers or heterozygotes (MZ, MS or M/Null) have levels approximately 35% of normal levels, but they do not develop disease.