Protein Turnover and Amino Acid Catabolism Stryer Ch. 23 Biochemistry Protein Turnover and Amino Acid Catabolism Stryer Ch. 23

Protein Turnover Proteins are constantly being degraded and resynthesized Location of protein degradation Proteins from diet are hydrolyzed in the digestive tract Proteins within each cell are broken down within that cell in a proteasome EXCESS AMINO ACIDS ARE NEITHER STORED NOR EXCRETED Degradation of amino acids requires Removal of amino group – deamination Urea cycle Conversion of carbon skeletons into glucose / glycogen via conversion to: Acetyl CoA Acetoacetyl CoA Pyruvate Citric Cycle intermediate Amino Acids are classified as essential and nonessential Essential – organism lacks the ability to synthesize these amino acids; must be acquire from diet Nonessential – amino acids are synthesized in vivo from smaller non amino acid precursors

Degradation of Dietary Proteins Dietary Proteins are digested by general and specific proteases into free amino acids, dipeptides and tripeptides, which are absorbed into the intestine by specific transporters. Stomach Acidic pH = 2 Pepsin – primary protease of stomach Lumen of Intestine MOST enzymes involved in digesting proteins are synthesized in the pancreas and secreted as zymogens. Aminopeptidase – nonspecific protease that sequentially hydrolyze proteins from the amino terminal end.

Degradation of Cellular Proteins Proteins turn over within the cell. Damaged / Mis-folded proteins are quickly destroyed Identified by attachment of ubiquitin Proteins that are no longer needed Half life of protein varies from protein to protein. Minutes, hours, days, years, decades. Determined by N-terminal residue R or L favors ubiquination (fast destruction) M or P disfavors ubiquination (slow destruction; long half life) Other signals exist. Some diseases are due to abnormal aggregation / improper destruction of proteins.

Ubiquitin (Ub) 8.5 kd protein Found in ALL eukaryotes “Mark of death” 76 amino acids Found in ALL eukaryotes “Mark of death” tags protein for proteolysis Ub covalently attached to proteins C-terminal gly of Ub Lys of protein ε amino group (R-group) Ub forms polymers polyubiquitination

Ub attachment requires three enzymes E1 or Ubiquitin-activating enzyme Activates Ub by attachment to AMP Links C-terminal carboxylate of Ub to sulfhydryl of E1 by thioester. E2 or Ubiquitin-Conjugating enzyme activated Ub transferred from sulfhydryl of E1 to sulfhydryl of E2 E3 or ubiquitin-protein ligase Catalyzes the transfer of Ub from E2 to target protein Largest gene family in humans

Disease linked to E3 Parkinson disease (some forms) Angelman syndrome Improper functioning E3 leads to abnormal accumulation of proteins. Angelman syndrome Severe neurological disorder (mental retardation, absence of speech, uncoordinated movement.) Human papilloma virus (HPV) Causes >90% of cervical carcinomas Activates specific E3 of host leading to destruction of p53 (tumor suppressor) and other DNA repair genes.

Structure of Proteasome 26 S Large protease complex Two 19S caps Contains AAA class ATPase activity ATPase associated with various activities One 20S catalytic core Four rings of 7 subunits each 28 subunits total 14 alpha (outer rings) Seven isoforms of alpha are known 14 beta (two inner rings) Seven isoforms of beta are known. Active sites are on beta subunits.

Action of Proteasome Hydrolyzes Ubiquinated proteins. Proteolytic active sites are located on the beta subunits Threonine residue acts as nucleophile to attach carbonyl of peptide bond. Digest proteins to 7 – 9 amino acid peptides These peptides are released from proteasome and further degraded to amino acids by cellular proteases.

Bortizomib (Velcade) Inhibitor of proteasome Therapy for multiple myeloma

Nitrogen Removal from Amino Acids Liver is primary site of amino acid degradation Muscle is secondary site of amino acid degradation for branched chain amino acids (e.g. L,I,V) Two step process

Removal of Amino Groups Aminotransferase (AKA transaminases) α amino group transferred to α-ketoglutarate to form glutamate Examples Aspartate amino transferase Alanine amino transferase Reversible Also used in synthesis of amino acids. Contain pyridoxal phosphate (PLP) as prosthetic group Derived from pyridoxine (vitamin B6) Glutamate dehydrogenase Converts nitrogen of glutamate to ketoacid and free ammonium ion Localized to mt of Liver May use EITHER NAD+ or NADP+ Close to equilibrium in liver Direction of reaction determined by [substrate] or [product] Normally driven forward by removal of ammonium

Pyridoxal Phosphate (PLP) Prosthetic group Group transfer to or from an amino acid. Precursor = Pyridoxine (Vitamin B6) Deficiency = depression, confusion, convulsions.

Pyridoxal Phosphate (Cont’) Mechanism see p 659 – 660.

Serine and Threonine are directly deaminated Serine dehydratase dehydrates and deaminates serine to produce pyruvate and ammonium Amino group is NOT transferred to α-ketoglutarate Threonine dehydratase dehydrates then deaminates threonine to yield α-ketobutyrate and ammonium

Peripheral Tissues Transport Nitrogen to the Liver Glucose – Alanine Cycle Muscle tissue uses branched amino acids as fuel. The nitrogen from these amino acids is transferred to alanine (through glutamate). Alanine is carried to liver via blood stream. Alanine is converted to pyruvate which is used to produce glucose.

Excretion of Nitrogen Terrestrial vertebrates Ureotelic – excrete excess nitrogen as urea Aquatic vertebrates and invertibrates Ammoniotelic – excrete excess nitrogen as ammonium Birds and reptiles Uricotelic – excrete excess nitrogen as uric acid

Urea Cycle Hans Krebs and Kurt Henseleit (1932) Cycle responsible for synthesis of urea Urea = form of nitrogen excreted in vertebrates. Humans excrete 10 kg urea / yr. 10 kg = 22 lbs. Source of atoms in Urea 1 N from free NH4+ ammonium C from HCO3- bicarbonate derived from hydration of CO2 1 N from aspartate Carbamoyl Phosphate intermediate in urea cycle Synthesized from NH3 and HCO3- Carbamoyl group has a high phosphate transfer potential due to anhydride bond.

Carbamoyl Phosphate Synthetase Catalyzes the three step synthesis of carbamoyl phosphate Bicarbonate phosphoryled by phosphate from ATP forming carboxyphosphate. Carboxyphosphate reacts with ammonia to form carbamic acid. Carbamic acid is phosphorylated by ATP to yield carbamoyl phosphate. Mt matrix 2 ATP / carbamoyl phosphate synthesized Isozyme catalyzes the synthesis of carbamoyl phosphate for use in pyrimidine biosynthesis

Carbamoyl Phosphate Synthetase Three reaction sites Glutamine hydrolysis site Bicarbonate phosphorylation site Carbamic acid phosphorylation site Substrate channeling Substrates pass from one active site to another active site through a channel. Is not released by enzyme. Benefits Increases the rate of reaction because the substrate is not “released” from the enzyme. Protects libile substrates from degradation by hydrolysis Carbonic acid decomposes in 1 s at ph =7.

Ornithine transcarbamoylase Catalyzes the synthesis of citrulline from ornithine and carbamoyl phosphate Citrulline and ornithine Amino acids NOT used in synthesis of proteins. Mt matrix Following synthesis, citrulline is transported to cytoplasm

Argininosuccinate Synthetase Catalyzes the condensation of citrulline with aspartate to form argininosuccinate Cytoplasm 1 ATP / argininosuccinate synthesized Hydrolized to AMP and PPi

Argininosuccinase Cleaves argininosuccinate into arginine and fumarate. Conserves carbon skeleton of aspartate in fumarate. Cytoplasm

Arginase Hydrolyzes arginine into ornithine and urea. Ornithine transported into mt Urea excreted.

Urea Cycle is Linked to Gluconeogenesis Aspartate and Fumarate link Urea Cycle to Gluconeogenesis.

Inherited Diseases of the Urea Cycle Hyperammonemia Elevated levels of ammonium (NH4+) in blood Causes include: lack / reduced synthesis of carbamoyl phosphate Lack / reduced activity of the four enzymes of urea synthesis Why is excess ammonium toxic? Maybe the synthesis / accumulation of glutamine causes an osmotic imbalance???

Treatments of Hyperammonemias Argininosuccinase deficiency treatment Provide excess arginine in diet Restrict total protein in diet reduces amount of nitrogen to be excreted Arginine is converted into Ornithine which reacts with Carbamyol phosphate to form citrulline. Citrulline reacts with aspartate to form arginiosuccinate which is excreted.

Treatments of Hyperammonemias (cont’) Carbamoyl phosphate synthetase or ornithine transcarbamoylase deficiency treatment Excess nitrogen accumulates in glycine and glutamine. Restrict protein in diet Supplement diet with benzoate and phenylacetate Converted into Benzoyl CoA and phenylacetate CoA Excess nitrogen excreted as hippurate or phenylacetylglutamine

Fates of Carbon Skeletons Carbon skeletons from deaminated amino acids are converted into seven intermediates Pyruvate Acetyl CoA - K Acetoacetyl CoA - K α-Ketoglutarate Succinyl CoA Fumarate Oxaloacetate Ketogenic Amino Acids Carbon skeletons are converted into intermediates (acetyl CoA or Acetoacetyl CoA) that can form ketone bodies or fatty acids Glucogenic Amino Acids Carbon skeletons are converted into intermediates that can be used to synthesize glucose. Both Some amino acids have carbons that end up in ketogenic and glucogenic intermediates.

Fates of Carbon Skeletons

Amino Acids GLUCOGENIC BOTH KETOGENIC NONESSENTIAL Alanine (A, Ala) Arginine (R, Arg)* Asparagine (N,Asn ) Aspartate (D, Asp) Cysteine (C, Cys) Glutamate (E, Glu) Glutamine (Q, Gln) Glycine (G, Gly) Proline (P, Pro) Serine (S, Ser) ESSENTIAL (Val and His Three Methods) Histidine (H, His)* Methionine (M, Met) Threonin (T, Thr) Valine (V, Val) BOTH KETOGENIC NONESSENTIAL NONESSENTAIL Tyrosine (Y, Tyr) ESSENTIAL ESSENTAIL (Iley Trpped BOTH Phesants) KETONES in Leu of Lysine Isoleucine (I, ile) --Leucine (L, Leu) Phenylalanine (F, Phe) -- Lysine (K, Lys) Tryptophane (W, Trp) Essential: TV FILM HW(R)K Updated 2014

Amino Acids GLUCOGENIC BOTH KETOGENIC NONESSENTIAL A R * N D C E Q G P (Val and His Three Methods) H, His* M, Met T, Thr V, Val BOTH KETOGENIC NONESSENTIAL NONESSENTAIL Y ESSENTIAL ESSENTAIL (Iley Trpped BOTH Phesants) KETONES in Leu of Lysine I, Ile --L, Leu F, Phe --K, Lys W, Trp Essential: TV FILM HW(R)K Updated 2014

Definitions Glucogenic Amino Acids Ketogenic Amino Acids Carbon skeletons are converted into intermediates that can be used to synthesize glucose. Ketogenic Amino Acids Carbon skeletons are converted into intermediates (acetyl CoA or Acetoacetyl CoA) that can form ketone bodies or fatty acids NOT substrates for glyconeogenesis Nonessential Amino Acids enzymes present for de novo synthesis of these amino acids Essential Amino Acids lacks enzymes to synthesize the amino acids. Must be obtained from diet.

Pyruvate Amino Acids with 3 C backbone Ala, Trp Ser, Gly Cys Thr Sulfur is converted to H2S, SCN- or SO32- Thr 2-amino-3-ketobutyrate intermediate

Acetyl CoA Acetoacetyl CoA Branched chain (Leu, Ile, Lys,) and aromatic amino acids (Phe, Trp, Tyr)

Oxygenases are Required for Degradation of Aromatic Amino acids Phenylalanine hydroxylase Hydroxylates Phe to Tyr Monooxygenase Each atom of oxygen incorporated into different products Tyr and water Tetrahydrobiopterin Electron carrier

Tetrahydrobiopterin (BH4) Electron carrier Derived from Biopterin Synthesized in vivo from phenylalanine NOT a vitamin because it is synthesized by the body. Phenylalanine hydroxylase Synthesizes dihydrobiopterin by hydroxylating phenylalanine Dihydrofolate reductase Reduces dihydrobiopterin to tetrahydrobiopterin using NADPH Dihydropteridine reductase Reduces quinonoid dihydrobiopterin itto Tetrahydrobiopterin using NADPH

Phe / Tyr 1.Transamination 2. p-hydroxyphenylpyruvate hydroxylase Dioxygenase both atom of oxygen are incorporated into the product 3. Homogentisate oxidase 4.Isomerization 5. Hydrolyzed 2 3 1 4 5

Trp Converted into alanine and acetoacetate via the action of several oxidases. 1 2 3 4

α-Ketoglutarate Amino acids with C5 skeletons Glu Gln Pro Arg His Deaminated into α-Ketoglutarate Gln Hydrolyzed to glutamate by Glutaminase Pro Converted to glutamate γ-semialdehyde Arg His Multistep process with 4-imidazolone 5-propionate intermediate

Succinyl CoA Major intermediates are common to oxidation of odd numbered fatty acids. Propionyl CoA Methylmalonyl CoA Succinyl CoA Met Nine steps involving S-adenosylmethionine (SAM) as methyl donor. See next slide Val Iso

Met degradation

Fumarate Asp Tyr Some carbons Phe

Oxaloacetate Asp Asn Direct deamination into Oxa NH4+ removed by Asparaginase to form Asp which is deaminated

Alcaptonuria Archibald Garrod (1902) First description of an inborn error of metabolism Defective degradation of phe and tyr. Recessive inheritance Homogentisate accumulates and is excreted in the urine. Harmless condition

Maple Syrup Urine Disease (branched-chain ketoaciduria) Defective / missing branched-chain dehydrogenase Defective degradation of val, ile, leu. Causes elevation of the level of these amino acids and the α ketoacid derivatives in vivo and in urine. Causes Mental and Physical retardation Patients urine smells like maple syrup. Detection Reaction of α ketoacid in urine with 2,4-dinitrophenylhydrazine Mass Spec.

Phenylketonuria (PKU) Defective / missing phenylalanine hydroxylase (or the tetrahydrobiopterin cofactor) Phenylalanine cannot be converted into tyrosine therefore phe accumulates Autosomal recessive Due to high [phe], reactions of Phe not found in normal individuals are prevalent in PKU patients e.g. formation of phenylpyruvate Phenylpyruvate in urine with FeCl3 to turn urine olive green. (this reaction lead to the initial description of PKU) Untreated PKU leads to retardation and reduced like expectancy (death in 20 – 30s). Treatment Low phenylalanine diet. Just enough phe for growth and replacement. Prevents accumulation of phe Must be started soon after birth.

Other Diseases involving Enzymes of Amino Acid Degradation