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AA and Proteins Robert F. Waters, PhD Overview Proteins – Structural – Enzymatic Amino Acids Henderson-Hasselbach Equation Acidity and Alkalinity Gas exchange Proteins Polypeptides with peptide bonds – Peptide bonds » Endergonic (Consume energy) Need energy and do not occur spontaneously Structural proteins Soluble proteins (Enzymes) Protein Structure Primary protein structure – Sequence of amino acids » Nomenclature: ala-glu-gly (N-terminus to C-terminus) alanylglutamylglycine Secondary structure – -helix and -sheet Tertiary structure – 3-dimensional folding Quaternary structure – Multiple subunits of tertiary structures Protein Structure: Primary Amino terminus Carboxyl terminus Protein Structure:Secondary Protein Structure: Tertiary and Quaternary Forces That Stabilize Proteins Ionic bond Hydrogen bonding Hydrophobic interactions – Hydrocarbons in aqueous solution have force association with adjacent hydrocarbons by rearrangement of surrounding water molecules Van der Waals interactions – Weak electrostatic attractions Denaturation of Proteins Soluble Proteins Precipitate Dehydration Heat Radiation pH Cold Pressure Chemicals Excessive vibrational energy (Microwaves) Natural organic substances (e.g., urea) Reducing agents – e.g., Mercaptoethanol HS-CH2-CH2-OH » Blocks disulfide bond formation Post-Translational Denaturation Associated with Golgi Apparatus – Packaging – Folding Herbicides Pesticides Neurotoxins (snake venom) Example of Precipitation by Acidification Milk proteins (Two Main Types) – Casein » 1-casein, s2-casein, -casein, -casein, -lactalbumin, -lactoglobulin – Whey (serum protein) » Serum albumen, immunoglobulins, lactoferrin Casein separated from whey by acidification to casein pI of 6.0. – Like adding citrus to coffee with cream » Serum (whey)proteins remain in solution while casein precipitates – Casein with lipids forms micelles (opaqueness of milk) Whey protein (hydrophilic) is used as protein addition to drinks, thickeners Casein is an excellent emulsifier in the addition of flavoring agents Vitamins and Minerals May Give Color to Protein When a vitamin or mineral gives a protein color is called a chromophore FAD or FMN added to apoproteins to form flavoproteins give a yellowish color Iron with myoglobin in meat – Ranges in color from brown to bright red – White poultry meat has low myoglobin – Dark meat has high myoglobin content – Veal and pork have less myoglobin than beef Myoglobin and hemoglobin without iron are colorless Myoglobin and hemoglobin with iron are pink to red Cooking meat dissociates heme to protein, iron and other complexes and produces brown to tan color Quantifying Protein in Solution Based on absorption spectra of aromatic amino acids (~280nm) – Tryptophan, tyrosine, phenylalanine – Different proteins may vary in aromatic amino acids but absorption spectra variation is still useful Zymogen System Series of enzyme activations for the digestion of protein into amino acids Protection mechanism against autolysis of endogenous proteins Begins mainly in the stomach and proceeds to intestine The Stomach:Overview Stomach not a very absorptive organ but— – Water, ETOH, short and medium chain FAs are absorbed Gastric mucosa – Chief cells, parietal cells and mucous cells » Produce gastric juices called gastrins Summary of Stomach Gastrins Parietal Cells of Stomach – Secrete HCL » Denaturation, very little digestion – Secrete Vitamin B12 intrinsic factor Chief Cells of Stomach – Secrete gastric lipase – Secrete pepsinogens » Activated to pepsin by HCL » Activated to pepsin by sutolysis Pepsin cleaves proteins into large oligopeptides (peptones) Mucosa cells – Secrete bicarbonate and mucus Stomach Summary Cont: Stimulation of gastric secretions (Gastrin) – – – – Protein itself Vagal stimulation Calcium ions Alkalination of the stomach Gastrin stimulates – – HCL production (parietal cells) » HCL inhibits gastrin production » NaCl necessary for HCL production – Mucin (mucous cells) – Pepsinogen production (chief cells) » Activated by HCL to Pepsin Zymogens Proenzymes (zymogens) packaged as zymogen granules in pancreas Pancreatic zymogens are serine proteases – Trypsin (less than proenzyme form) – Chymotrypsin – Elastase Enteropeptidase Activation Enteropeptidase produced in intestinal brush border – Activates trypsin from trypsinogen » Some activation of trypsin by autolysis » Trypsin activates chymotrypsinogen, elastase and carboxypeptidase A and B (possibly some aminopeptidases) Carboxypeptidases and Aminopeptidases are called Exopeptidases Graphic RepresentationEnteropeptidases and Cascading Phosphorous Containing Nerve Gases Initial studies with acetylcholine esterase Nerve gas DFP – Diisopropylfluorophosphate – Attacks serine hydroxyl groups in enzymes like acteylcholine esterase AND serine proteases like chymotrypsin » Attacks serine 195 in chymotrypsin DFP acts as a pseudo-substrate for the enzymes DFP stops enzymatic reaction Graphical Representation of DFP Cysteine Proteases Attack sulfhydryl group on cysteine in protein Examples (mammals have similar proteases) – – – – – Papain (papaya) Bromelain (pineapple) Ficin (fig) Actinidin (kiwi fruit) Caricain, chymopapain, glycyl endopeptidase (from latex portion of papaya tree) Lysosomal Proteases Active at lower lysosomal pH Cathepsins – Cathepsin B (Most abundant) » Endopeptidase and Exopeptidase – Cathepsin H (Aminopeptidase) – Cathepsin K (Abundant in bone resorbing osteoclasts » Absence causes fragile small bones – Cathepsin C (dipeptidyl peptidase) » Removes N-terminus dipeptides activating intracellular proteins and maybe other Cathepsins Bleomycin hydrolase – Bleomycin is an anti-cancer drug – Bleomycin hydrolase breaks down bleomycin » Unfortunately cancer cells have high amounts of this enzyme causing drug resistance – Papain-like activity that also binds to DNA? Cysteinyl Aspartate-Specific Proteases (Caspases) Involved in programmed cell death (Apoptosis) Activation of Interleukins – Caspase-1 (AKA: Interleukin-1-converting Enzyme or ICE) » Cleaves pro-interleukin-1 to form the active interleukin-1 Aspartate Proteases (Pepsin-Like) Example is gastric proteinase – Gastricsin – Has similar activity as rennin (chymosin) from the fourth stomach of calf » Causes rapid clotting of milk Used in cheese manufacturing – Serum protein Renin (NOT rennin) is similar as well Protease Inhibitors-Exogenous Leupeptin (Inhibits trypsin) Boronic Acids (Inhibit serine proteases) Pepstatin (Inhibit aspartic proteases) – Were “Lead Compounds” for the formation of HIV protease inhibitors Mercaptans (Inhibit Zn++ metalloproteases) – Bind to Zn++ in some metalloproteases » Captopril Drug that inhibits Angiotensin-Converting-Enzyme (ACE) Endogenous Protease Inhibitors Trypsin activation in pancreas would be disastrous – Pancreatic Trypsin Inhibitor Serpins (Blood) – Inhibit serine proteases – 10% of the total protein in blood » 1-protease Inhibitor (1-antitrypsin) Found in -globulin fraction of blood » NOTE:One form of emphysema is the hereditary absence of 1-antitrypsin Without this inhibitor, tissue will degrade excessively, e.g. elastin, collagen and proteoglycans Protease Activities-Serine Proteases Amino Acids Last one described was threonine in 1938 Stereospecific (L-Configuration exclusively) – D-Amino acids found in bacteria Chemical properties associated with stereospecificity and side groups Easily ionized in aqueous solution Produces a zwitterion (dipolar chemical structure with + and – charges Zwitterion effect causes crystalline form of amino acids to have high decomposition temperatures above 200o centigrade – Similar to electrostatic forces holding and NaCl lattice together Overall Structure of Amino Acids -Carbon, Carboxyl Group, Amino Group – Except imino amino acids Enantiomeres (L-Amino acids in proteins) – D (Dextro) and L (laevo) Amino Acid Structures-Neutral Side Groups Amino Acid Structures-Aromatic and Acidic Side Chains Amino Acid Structures-Positive Side Groups Amino Acid Structures-Polar Side Groups Classification by Polarity Essential Amino Acids PVT TIM HALL Phenylalanine, valine, trptophan, threonine, isoleucine, methionine, histidine, arginine (neonate-child), leucine, lysine Non-Essential Amino Acids Synthesized by humans – Serine, glycine, cysteine, alanine, aspartate, asparagine, glutamate, glutamine, proline, arginine (adult), tyrosine (from phenylalanine) Non-Protein Amino Acids Citrulline is a product of L-arginine synthesis (urea cycle) and NO (Nitric Oxide) metabolism Creatinine is derived from muscle – Plasma amounts to muscle mass Ornithine, taurine, homocysteine Biogenic Amine Compounds – Dopamine, serotonin, histamine Amino Acids and pH pH = -log10 [H+] ion concentration – Alkalinity vs. acidity Absorption of AA and pH Henderson-Hasselbach Equation (H-H) HA is protonated form – Conjugate acid or associated form A- is unprotonated form – Conjugate base or dissociated form Protonation occurs in acidic solutions Removal of protons in more alkaline solutions H-H Continued: Acidic amino acids – Glutamate and aspartate are negatively charged acidic amino acids at physiological pH Basic amino acids – Arginine, lysine, and histidine are positively charged amino acids at physiological pH pK Values for Amino Acids Titration of Amino Acids with (OH ) NaOH-Glycine Titration of Amino Acids with (OH ) NaOH-Histidine Aspects of Titration pI = Isoelectric Point – pH where net charges equal zero (0) » Histidine pI = 7.7 (6.0 + 9.3)/2 = 7.7 » pHm = (1.8 + 6.0)/2 = 3.9 pHm = Maximum Charge – pH where number of positive and negative charges are maximal Buffering Range – Range where change in pH is minimal – Approximately +1 pH above pK to –1 pH below pK value » e.g. if a pK value = 6.0, then the buffering range around this pK would be 5.0 – 7.0 Importance of Regulating pH Denaturation of Protein – Enzymes » Charge distribution » Hydrolysis of bonds Charge changes on amino acids – Substrate specificity Hydroxyl amino acids – Serine, threonine, tyrosine Quaternary structure – Proteins, hemoglobin binding of oxygen pK of amino group – Free amino acid amino group pK = 9.5 – Amino group in a polypeptide pK = 8.0 Extremes of pH Acidosis to Alkalosis – Acidotic condition pH=7.0 – Alkalotic condition pH=8.0 Normal pH = 7.4 – Venous blood pH = 7.35 – Arterial blood pH = 7.45 » Higher altitude arterial pH = 7.49 Most extreme limits are pH = 7.0 – 8.0 Acidosis Acidosis – Hyperventilation » Controlled by mid brain – Acidotic coma » Reduction in myocardial contractions » Reduction in catacholamines (e.g., histamine) Reduce vascular tone (shock) Pumping blood against a non-resistant wall » Hypooxygenated (Cannot bring in enough O2) Excess protons block hemoglobin binding of O2 – “Bohr Effect” Acidosis Continued: Sources of Protons – Volatile acids (Respiratory) – First conversion is carbonic anhydrase – Second reaction is spontaneous 3 CO2 H 2O H 2CO3 HCO H – Non-volatile acids » Lactate (Metabolic) » Ketones (Liver produces these thinking there is a lack of glucose) » Sulfuric (From Cysteine Degradation) Alkalosis Alkalosis – Hypoventilation – Tetany (Sustained uncontrolled muscle contraction) » Muscle contraction controlled mainly by Na in neuron and Ca in muscle 2 H P O HPO » Related to phosphate 2 4 4 Monobasic and dibasic forms Under alkalosis with the addition of [OH] you form water Also drive reaction to the right H 2 P O4 HPO42 H OH H Alkalosis Continued Dibasic form of phosphate can chelate soluble calcium better than monobasic form to form calcium phosphate 2 4 H 2 P O4 HPO H Does not change blood concentration of calcium only amount that is soluble Alkalosis Continued What does calcium do in nerve impulses? – Inhibits transport of sodium into nerve during the process of depolarization due to a nerve impulse – Under alkalotic conditions more calcium is chelated and removes the controlled blockage of sodium into nerve (uncontrolled influx of sodium into nerve) » More nerve depolarization Causes the sustained uncontrolled muscle contraction during alkalosis (tetany) Air Exchange Partial Pressures of Gases in Air – 80% N2 – 20% O2 – .03% CO2 1 atm = 760mm Hg (Sea level) Example: – pN2 = .79 * 760 = 608mm Hg – pO2 = .20 * 760 = 152mm Hg – pCO2 = .0003 * 760 = 0.23mm Hg Gas Exchange pCO2 of blood is around 40mm Hg pCO2 in lungs is around 35mm Hg Partial pressures of respiring cells higher yet