Nitrogen Fixation & Incorporation Lecture 2024 PDF

Nitrogen Fixation & Incorporation Wednesday October 23, 2024  Goals  Recognize different nitrogen containing molecules  Understand definitions for nitrogen fixation and nitrogen assimilation  Describe the reactions catalyzed by the nitrogenase com...

Nitrogen Fixation & Incorporation Wednesday October 23, 2024  Goals  Recognize different nitrogen containing molecules  Understand definitions for nitrogen fixation and nitrogen assimilation  Describe the reactions catalyzed by the nitrogenase complex, glutamine synthetase, glutamate synthase, glutamate dehydrogenase and transaminases/aminotransferases  Understand definitions of essential and nonessential amino acids  Define nitrogen balance, +ve nitrogen balance and –ve nitrogen balance Why is nitrogen important? Know your nitrogen N2 NH3 NH4+ nitrogen gas ammonia ammonium NO2- NO3- nitrite nitrate Nitrogen gas Air is ~78% N2 N2 is inert (stable/nonreactive) and we can’t use Atmospheric N2 must be reduced to a useful form Nitrogen fixation = reduction of atmospheric N2 - can only be done by some prokaryotes  Nitrogen Fixation H H N H H H N N H N N H N N + H H H N H H Reaction requires lots of energy!! N2+8e- + 16ATP + 10H+  2NH3 + 16ADP + 16Pi + H2 Overall Reaction of Nitrogen Fixation The nitrogenase complex Enzyme complex responsible for reduction of N2 to NH3 Two different proteins in the complex – dinitrogenase and dinitrogenase reductase (Fe containing proteins) Dinitrogenase reductase transfers electrons from NADH to dinitrogenase Dinitrogenase then reduces N2 Reaction is very slow – 6 per second Nitrogen assimilation Nitrogen assimilation – incorporation of inorganic nitrogen (e.g. NH4+ or NO3-) into organic molecules Inorganic nitrogen is incorporated into amino acids Organic nitrogen moves through the ecosystem primarily as amino acids Glutamine synthetase – catalyzes this reaction Glutamate + NH4+ Glutamine This reaction is ATP dependent To incorporate nitrogen from NO3-, the nitrate must be reduced to ammonium first Glutamate Synthase Glutamine acts as nitrogen donor for reductive amination of a-ketoglutarate Catalyzed by glutamate synthase (only in prokaryotes and plants) Requires NADPH Enzyme is not present in animals a-ketoglutarate + glutamine + NADPH + H+ 2 glutamate + NADP+ Amino group from glutamate can be transferred in the synthesis of other molecules Glutamine Synthetase and Glutamate Synthase - recap O H O O H O O C CH2 CH2 C C O- - + NH +ATP 4 + H2N C CH2 CH2 C C O- +ADP+H + NH3+ NH3+ Catalyzed by glutamine synthetase O H O O H O O O O - O C CH2 CH2 C C O- H2N C CH2 CH2 C C O- + O C CH2 CH2 C C O- - + NADPH +H NH3+ NH 3 + O O Catalyzed by glutamate synthase - O C CH2 CH2 C C O- + NH3+ NADP+  Direct amination of a-ketoglutarate Glutamate dehydrogenase catalyzes this reaction Reaction is reversible Reaction primarily runs in reverse in eukaryotes – to generate NH4+ for nitrogen excretion Very minor mechanism to assimilate nitrogen O O O O H O O C CH2 CH2 C C O- - +NH +NADH 4 + O C CH2 CH2 C C O- NAD+ - + NH3+ Catalyzed by glutamate dehydrogenase Amino Acid Biosynthesis Plants and prokaryotes can make all of their amino acids Animals can only make some amino acids (nonessential amino acids) and must get the other amino acids from their diet (essential amino acids)  Humans cannot make these three amino acids biosynthetically, but they can be produced as a byproduct of other biochemical pathways Table 14.1 The Essential And nessential Amino Acids In Humans TRUDY MCKEE, JAMES R. MCKEE, Biochemistry, The Molecular Basis of Life 13 © 2020 Oxford University Press Nitrogen balance When nitrogen uptake = nitrogen excretion, nitrogen is in balance Positive nitrogen balance - nitrogen uptake > nitrogen excretion Can occur in children, i.e. they are growing Negative nitrogen balance - nitrogen uptake < nitrogen excretion Can result in malnutrition, e.g. Kwashiorkor Amino Acid Metabolism Principle source of nitrogen for biosynthetic reactions Source of amino acids is the diet Metabolic mechanisms are required since dietary amino acids may not be in the proportions that we need Recall gluconeogenesis BCAA (branched chain amino acids) – transport of nitrogen from liver to other tissues Amino acids cross cell membranes using specific transport proteins Overview of reactions in amino acid and nucleotide biosynthesis Three major types of reactions we will discuss Transamination reactions catalyzed by pyridoxal phosphate containing enzymes – aminotransferases or transaminases Transfer of one carbon groups using THF or SAM cofactors Transfer of amino groups from glutamine Transamination reaction H O O O O O H O R1 C C O- +R 2 C C O- R1 C C O- +R2 C C O- NH3+ NH3+ Donor Acceptor New New amino acid keto acid keto acid amino acid Catalyzed by aminotransferases or transaminases Transamination reactions Reversible reactions Function in synthesis and degradation of amino acids Aminotransferases – many in eukaryotes Cytoplasmic and mitochondrial enzymes Specifically recognize the donor amino acid and acceptor keto acid Glutamate is most often donor amino acid – α-ketoglutarate/glutamate pair is important Oxaloacetate/aspartate pair – important for urea cycle (disposal of nitrogen) Pyruvate/alanine pair important in glucose-alanine cycle (gluconeogenesis) Pyridoxal-5’-phosphate Transamination reaction requires a co-enzyme – pyridoxal-5’-phosphate (PLP) Vitamin B6 PLP binds the active site of transaminases from different sources PLP is coenzyme for racemization reactions PLP is coenzyme for decarboxylation reactions Biologically active form of vitamin B6 is PLP PLP forms a Schiff base PLP PLP bound to enzyme as Schiff base PLP forms a Schiff base with donor amino acid Break this bond Form new covalent bond To make An imine exchange reactions this product results in Schiff base formation between PLP and amino acid FIGURE 6 i PLP forms a Schiff base with donor amino acid Breaking this bond removes the N from the donor amino acid 3 bonds could potentially be broken - Bond 1 results in decarboxylation - Bond 2 results in transamination - Bond 3 results in racemization FIGURE 5 Intermediate Schiff Base Formed between Pyridoxine and an Amino Acid  Break this bond Amino group from donor amino acid New keto acid FIGURE 6 iii  * Generate this product Form new covalent bond Acceptor keto acid * FIGURE 6 iii R* is different than R  PLP forms a Schiff base with donor amino acid New amino acid * * Break this bond An imine exchange reaction This carbon forms results in Schiff base formation covalent bond with between PLP and the enzyme enzyme FIGURE 6 i  Keto acid now leaves the active site PLP – covalently bound to NH2 remains in active site Acceptor keto acid now binds to active site Reverse of the reactions results in transfer of the NH2 group from PLP to the acceptor keto acid This mechanism is a ping-pong mechanism or double displacement mechanism