What to Know (protease lecture)

1. What to Know (protease lecture) • Know the general mechanism  of serine proteases • what imparts specificity? • how is the substrate stabilized? • how is the transition state stabilized? • what amino acids play key roles and why? • Understand the major biological roles of the four different proteases mentioned in the lecture

2. Nitrogen Degradation

3. Things to Know • What purpose does nitrogen fixation and assimilation serve in the biosphere? • What are the key enzymes in nitrogen fixation and assimilation? • What are examples of nitrogen fixation and assimilation in life? • What are the generalconcepts for the pathways that form ammonium from inorganic nitrogen compounds prevalent in the inanimate environment? • What are the general concepts of how ammonium ions are incorporated into organic compounds? • What are the general concepts of how amino acids are synthesized and degraded?

4. Nitrogen Cycles • Nitrogen in proteins is reduced • Inorganic nitrogen in the environment is oxidized as nitrogen gas or nitrate ions • Two principal routes for nitrogen acquisition from the environment lead to formation of NH4+ • Nitrate assimilation • nitrogen fixation • Are animals capable of nitrogen or NO3- fixation?

5. Nitrogen is cycled between organisms and the inanimate environment • Nitrate assimilation – the reduction of nitrate to NH4+ in plants, various fungi, and certain bacteria, in a two-step metabolic pathway • Nitrogen fixation – the formation of NH4+ from N2 gas

6. Major Pathways for N Acquisition Denitrifying bacteria: Exclusively anaerobic, use NO3- as electron acceptors Exclusively anaerobic, prokaryotic process except for bacteria in symbiotic relationship with green plants Nitrate Assimilation aerobic process Nitrifying bacteria chemoautotrophs

8. Nitrate assimilation – the reduction of nitrate to NH4+ in plants, various fungi, and certain bacteria, in a two-step metabolic pathway • Nitrate assimilation occurs in two steps: • The two-electron reduction of nitrate to nitrite, catalyzed by nitrate reductase • NO3- + 2H+ +2e-nitrate reductase NO2- + H2O • The six-electron reduction of nitrite to ammonium, catalyzed by nitrite reductase • NO2- + 8H+ + 6e- nitrite reductase NH4+ +2H2O

9. Nitrate Assimilation Nitrate Reductase contains cytochrome b557 and molybdenum cofactor (MoCo)

10. Nitrate Reductase

11. Nitrate Assimilation is the Principal Pathway for Ammonium Biosynthesis Prosthetic Group of Nitrate Reductase

12. The Reaction of Nitrite Reductase Prosthetic Group of Nitrite Reductase act as coupled e- transport center

13. Nitrite Reductase

14. Nitrate Assimilation is the Principal Pathway for Ammonium Biosynthesis

15. Organisms Gain Access to Atmospheric N2 Via the Pathway of Nitrogen Fixation Only occurs in certain prokaryotes • Bacteria can use nitrogen fixation reactions to convert atmospheric nitrogen into ammonium ions. • N2 fixing bacteria can be free-living or as symbionts with plants.

16. Nitrogen Fixation • All nitrogen fixing systems appear to be identical-- They require 4 key components: • The enzyme known as Nitrogenase, • a reductant (reduced ferredoxin), • ATP • O-free conditions and regulatory controls (ADP inhibits and NH4+ inhibits expression of nif genes)

17. The Nitrogenase Reaction N2 + 10H+ 8e- 2NH4+ + H2 Two protein components: nitrogenase reductase and nitrogenase

18. Nitrogenase Complex Ribbon diagram of nitrogenase reductase (the Fe-protein, blue); nitrogenase (FeMo) protein, green) complex. Iron-sulfur cluster is yellow, ADP in orange, FeMo in cyan, P=cluster in red.

19. Organisms Gain Access to Atmospheric N2 Via the Pathway of Nitrogen Fixation The triple bond in N2 must be broken during nitrogen fixation.

20. The Metal Clusters of Nitrogenase

21. The Nitrogenase Reaction • The nitrogenase reaction. ATP hydrolysis coupled to electron • transfer from N. reductase to P-cluster. • This is followed by conformational change in N. reductase so it does not bind to Nitrogenase. • ADP-N. reductase dissociates allowing another ATP-N. reductase to bind.

22. The Structure of Nitrogenase

25. Regulation of Nitrogen Fixation

26. Key Concepts / Amino Acid Metabolism • The enzymes glutamate synthase, glutamine synthetase, glutamate dehydrogenase, and aminotransferases are responsible for the vast majority of nitrogen metabolizing reactions in most organisms. • Protein degradation by the protozomal complex releases oligopeptides that are degraded into individual amino acids. • The urea cycle uses protons and electrons from ammonium ions and the amino acid aspartate to generate urea, which is excreted to maintain daily nitrogen balance.