BI/CH 422/622 OUTLINE: Protein Degradation (Catabolism) Digestion Inside of cells Protein turnover Ubiquitin Activation-E1 Conjugation-E2 Ligation-E3 Proteosome Amino-Acid Degradation Ammonia free transamination-mechanism to know Urea Cycle 5 Steps Carbamoyl-phosphate synthetase Ornithine transcarbamylase Arginino-succinate synthetase Arginino-succinase Arginase Energetics Urea Bi-cycle
Amino Acid Catabolism: Transamination
Vitamin B6
Structure of Pyridoxal Phosphate and Pyridoxamine Phosphate • Intermediate, enzyme-bound carrier of amino groups • The linkage is made via a nucleophilic attack of the amino group of an active-site lysine. • Making an internal aldimine
• Aminated form can react reversibly with carbonyl groups to make a Schiff base. • Aldehyde form can react reversibly with amino groups to make a Schiff base.
1 Amino Acid Catabolism: Transamination
Amino Acid Catabolism: Transamination
Aromatic Quinoid
2 Amino Acid Catabolism: Transamination
See Sapling animated Figure(a)
Aromatic Quinoid
http://media.saplinglearning.com/priv/he/lehninger/aem/1806a_pyridoxal_phosphate.html
Amino Acid Catabolism: Urea Cycle
Synthesis of Carbamoyl Phosphate
①
②
UREA ⑤ CYCLE③
④ Carbon dioxide
3 Amino Acid Catabolism: Urea Cycle ① Synthesis of Carbamoyl Phosphate • The first nitrogen-acquiring reaction of the urea cycle
Carbamoyl Phosphate Synthetase I
• Excess CO2, ATP, and ammonia is present in liver mitochondria. This is where the activation of both waste products occur (the majority of the other urea-cycle reactions occur within the cytosol). • For step #2, in order to move to the cytosol, carbamoyl phosphate must condense with ornithine to create citrullene. This reaction releases the phosphate of carbamoyl phosphate into the mitochondrial matrix (so it does not deplete the proton motive force). Citrullene can then be transported to the cytosol.
Amino Acid Catabolism: Urea Cycle ① Synthesis of Carbamoyl Phosphate See Sapling animated Figure http://media.saplinglearning.com/priv/he/lehninger/aem/1811a_carbamoyl_phosphate.html Ammonia
Ammonia travels
Carbamate travels Carbamoyl Phosphate N-acetyl- glutamate
Carbamoyl Phosphate Synthetase II Carbamoyl Phosphate Synthetase I
4 Amino Acid Catabolism: Urea Cycle
The Urea Cycle
①
②
UREA ⑤ CYCLE ③ ④
Amino Acid Catabolism: Urea Cycle The Urea Cycle: Evidence for a cycle -Already known that Arg gives rise to Urea and Orn ⑤
-How do CO2 and NH3 get into Arg? -Enter H. Krebs, who studies metabolism using the Warburg apparatus -Urease (recently purified by Sumner in 1925) can hydrolyzed urea to
CO2 + 2 NH3; Could measure CO2 from urea by Warburg apparatus -Using liver slices, which amino acids gave rise to urea? -….. Most gave some -Then as a control, he tried Orn -Added Orn to prep: extraordinary occurrence of “catalytic” amounts of urea!! Orn, which is the product, will give rise to more urea than the Orn added at a rate of 7-30x more: “catalytic”
-In the library, Krebs looked for intermediates that might have CO2 &
NH3 stuck to Orn: citrulline -Dr. Wada in Japan had just published to purification of citrulline from watermelon. -Krebs attained 10 mg citrulline and added it…...same catalytic phenomenon!! First cyclic process! Dr. Kornberg: Lecture …...lead to lots of notariety 03.15.17 (22:40-31:10)- Krebs (8 min)
5 Amino Acid Catabolism: Urea Cycle
The Urea Cycle
①
②
UREA ⑤ CYCLE ③ ④
Amino Acid Catabolism: Urea Cycle
② Ornithine Transcarbamylase (OTCase)
Recall: Aspartate Transcarbamoylase (ATCase) (a3)2(b2)3