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

Nitrogen Degradation

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 general concepts 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?

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+

1. Nitrate assimilation 2. nitrogen fixation

• Are animals capable of nitrogen or NO3- fixation?

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

Major Pathways for N Acquisition

Nitrate Assimilation aerobic process

Denitrifying bacteria: Exclusively anaerobic, use NO3

- as electron acceptors

Exclusively anaerobic, prokaryotic process except for bacteria in symbiotic relationship with green plants

Nitrifying bacteria chemoautotrophs

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

Nitrate AssimilationNitrate Reductase contains cytochrome b557 and molybdenum cofactor (MoCo)

Nitrate Reductase

Nitrate Assimilation is the Principal Pathway for Ammonium Biosynthesis

Prosthetic Group of Nitrate Reductase

The Reaction of Nitrite Reductase

act as coupled e- transport center

Prosthetic Group of Nitrite Reductase

Nitrite Reductase

Nitrate Assimilation is the Principal Pathway for Ammonium Biosynthesis

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.

Nitrogen Fixation

• All nitrogen fixing systems appear to be identical-- They require 4 key components: 1. The enzyme known as Nitrogenase, 2. a reductant (reduced ferredoxin), 3. ATP 4. O-free conditions and regulatory controls (ADP

inhibits and NH4+ inhibits expression of nif

genes)

The Nitrogenase Reaction

N2 + 10H+ 8e- 2NH4+ + H2

Two protein components: nitrogenase reductase and nitrogenase

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.

Organisms Gain Access to Atmospheric N2 Via the Pathway of Nitrogen Fixation

The triple bond in N2 must be broken during nitrogen fixation.

The Metal Clusters of Nitrogenase

The Nitrogenase Reaction

• The nitrogenase reaction. ATP hydrolysis coupled to electrontransfer 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.

The Structure of Nitrogenase

Regulation of Nitrogen Fixation

• 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.

Key Concepts / Amino Acid Metabolism