2Amino Acid MetabolismLiver is the primary site for amino acid metabolism.Each amino acid has a pathway for catabolism and separate one for anabolism. Actually the pathways differ in different organisms.For mammals: Essential amino acids must be obtained from diet. Nonessential amino acids - can be synthesized .
4Exogenous Protein Digestion Most dietary protein is hydrolyzed to amino acids and small peptides in the stomach and intestine.In stomach and intestinal
5Digestive Proteases Stomach (pH ~ 1-2): Pepsin, Gastricin, Chymosin Intestine (pH ~ 8): Trypsin, Chymotrypsin, Carboxypeptidase, Elastase Enteropeptidase, AminopeptidasesEndogenous protein is also catabolized but by a method that differs from that above. Also, endogenous proteins have varying lifetimes.
6Endogenoous Protein Turnover Proteins exhibit continuous turnover (synthesis and degradation). Protein half-lives are from minutes to months, most are short. The amino terminal residue is a factor in selection of some protein. ornithine decarboxylase t½ ~11 min liver & plasma protein ~2-10 days muscle protein ~180 days collagen ~1000 daysIn eucaryotes, some proteins are targeted for degradation by a covalent attachment through lysine residues of the target protein to the C- terminus of ubiquitin, a small 76 residue peptide.
7Ubiquitin on LysineAttachment of a lysine in the target protein to the C-term glycine of ubiquitin via an isopeptide bond.The signal for protein death.
8Ubiquitin Attachment of ubiquitin to Lys requires three enzymes: E1. Ubiquitin-activating enzyme (uses ATP)E2. Ubiquitin-conjugating enzyme (assembles Ubiq., the target protein and E3).E3. Ubiquitin-protein ligase (forms the Gly-Lys bond).
9Mechanism of Attachment an acyladenylateMany isoforms of E3 exist. These select proteins for degradation.
10Ubiquitin ChainsSequential attachment of C-term Gly to Lys48 of another ubiquitin forms tetra-ubiquitin. This extended structure serves as an enhanced degradation signal.
11A ProteasomeA Proteasome is a large ATP dependent complex that hydrolyzes the ubiquitinated proteins.
12Degradation EventsThe core of b subunits contain the active sites and all have an N-term Thr.The a subunits on the ends serve as regulatory caps that block access to the active sites.
14Procaryotic vs Eucaryotic Procaryotes have a proteasome analog of that in eucaryotes but the function is unclear since ubiquitin has not been found. In procaryotes, all α subunits are identical and all β subunits identical whereas in eucaryotes these subunits exhibit a number of isoforms.Procaryotes do have a ubiquitin-like protein but it is is used in the synthesis of thiamine and not protein degradation.
16Procaryotic vs Eucaryotic For thiamine synthesis For protein degradation
17Amino Acid CatabolismFirst step amino acid catabolism is generally removal of the a-amino group.Carbon chains are then altered for entry into central pathways of carbon metabolism.The amino acids from either degraded proteins or from a dietary source can be used for the biosynthesis of new proteins.During starvation proteins are degraded to amino acids to support glucose formation.
18Methods for Removal of NH3 1. Transamination:amino acid + a-ketoglutarate a-ketoacid + Glu2. Glutamate dehydrogenase:Glu + NAD+ a-ketoglutarate + NADH + NH4+3. Direct deamination:Ser pyruvateHis urocanate (resonance driven)4. Amide hydrolaseGln or Asn Glu or Asp + NH4+
191. TransaminationA pyridoxal phosphate (PLP) mediated reaction. a-ketoglutarate, the normal a-keto acid used, forms glutamate.
20PLPThe aldehyde group forms a schiff base with a Lys on a transaminase enzyme.PLP enzymes are typically involved in: transamination, decarboxylation, or racemization.
21Transaminase Mechanism Lysyl linkage is displaced by an amino acid.
22Transaminase Mechanism Loss of a proton and formation of a different Schiff base.
272. Glutamate Dehydrogenase The requirement for NAD+ or NADP+ in this enzyme varies. Glu (from transamination) -- > a-ketoglutarate
283. Direct Deamination Serine Dehydratase (uses PLP in Ecoli). In other amino acids direct deamination is driven by extended conjugation.
294. Amide HydrolysisNH4+ is removed from asparagine and glutamine by the enzymes asparaginase and glutaminase.asparaginase asparagine > aspartate + NH4+glutaminase glutamine > glutamate + NH4+
30Disposition of NH4+The ammonium ion (which is toxic) formed by action of a transaminase and glutamate dehydrogenase (below) or other reactions, goes to the urea cycle (in the liver).TransaminaseGlutamate DH
31Transport of NH4+NH4+ is transported to the liver by either of two methods.1. Glucose-Alanine Cycle (next slide)2. Glutamineglutamine NH4+ + glutamate + ATP > glutamine + ADP + Pi synthetaseglutaminase glutamine > glutamate + NH4+
33NH4+ to UreaWaste nitrogen must be removed (a high conc. of ammonia is cytotoxic) Fish and many aquatic organisms excrete NH4+, Terrestrial vertebrates synthesize urea, Birds, reptiles synthesize uric acid.The liver processes NH4+ into urea using carbamoyl-phosphate synthetase I and enzymes of the urea cycle.
34Incorporation of NH4+ Requires carbamoyl phosphate synthetase. Carbamoyl phosphate synthetase I catalyzes removal of ammonia using the energy from ATP to form carbamoyl phosphate. This is the normal feeder for the urea cycle which is a liver pathway.Carbamoyl phosphate synthetase II uses glutamine as a source of ammonia again using the energy from ATP to form carbamoyl phosphate. This provides carbamoyl-P for pyrimidine synthesis.
35Carbamoylphosphate Synthetase I A mitochondrial enzyme, requires 2 ATP
36Urea Cycle Four reactions. One in the mitochondria. Three in the cytosol.
37Urea Cycle, Reaction 1Transfer of the carbamoyl group in the mitochondria.
38Urea Cycle, Reaction 2A citrulline:ornithine antiport moves citrulline to the cytosol. The second NH2 for urea comes from Asp. An adenylated citrulline intermediate gives PPi.
39Urea Cycle, Reaction 3Cleavage of fumarate, production of arginine
40Urea Cycle, Reaction 4Cleavage of urea from arginine gives urea. The ornithine is ready to begin a new cycle.
41Recycling to Aspartate Malate dehydrogenase occurs in mitochondria and in the cytosol.
42N-AcetylglutamateN-AcGlu is synthesized when NH4+ levels increase during amino acid catabolism.An activator of CPS I which provides substrate for the urea cycle.Glu is a product of Gln hydrolysis by CPS II in pyrimidine synth.An intermediate in ornithine synthesis.
43Structural similarity Blue - Ornithine transcarbamoylase from the urea cycleRed - Aspartate transcarbamoylase from pyrimidine synthesis
44Functional similarity Transfer of an amino group by incorporation of aspartate and elimination of fumarate occurs in both the urea cycle and pyrimidine synthesis.
45AA Carbon SkeletonsIn catabolism of their carbon skeletons, amino acids are referred to as being either glucogenic or ketogenic depending upon the structures of the degradation products.Glucogenic amino acids degrade to pyruvate or citric acid cycle intermediates which can feed gluconeogenesis.Ala, Cys, Gly, Ser,Asp, Asn, Glu, Gln Thr depends upon the pathway.
46AA Carbon SkeletonsKetogenic amino acids degrade to acetylCoA or acetoacetylCoA which can contribute to the synthesis of fatty acids or ketone bodies.Leu and Lys are the only two purely ketogenic amino acids.Some amino acids yield both glucogenic and ketogenic parts.Phe, Tyr, Trp, Ile Thr depends upon the pathway.
54Ile, Val and LeuThese non-polar, branched chain amino acids follow a similar sequence of three reactions:1. Transamination giving an a-ketoacid.2. Oxidative decarboxylation giving an acylCoA.3. AcylCoA dehydrogenase yields a,b unsaturation.The residue from Ile follows b-ox to give acetylCoA and propionylCoA.The residue from Val adds HOH, is then oxidized twice to yield an acid with a b-carbonyl. This decarboxylates to give propionylCoA.