2. Acetoacetate decarboxylase (AADase) is a classic example of an enzyme that significantly changes the pKa of a reactiveside chain by creating a microenvironment that favors one protonation state of this side chain vs the other protonation state.Below is the reaction catalyzed by AADase, which is important for acetone production during biosynthesis of ketone bodiesin mammals and solventogenesis in bacteria:CO2HO"CHз АADaseacetoacetateacetonea) AADase-catalyzed decarboxylation of acetoacetate proceeds through an enzymatic Schiff base. Draw out the mechanismfor this conversion of acetoacetate to acetone, showing enzymatic lysine (Lys, or K) involvement.b) Draw out the protonation/deprotonation equilibrium for Lys. What form of Lys is catalytic in part a: protonated ordeprotonated? If Lys pKa in AADase was the same as its pKa in solution, what would be the predominant form of Lys atphysiological pH? Which way would Lys pKa need to change (if at all) for efficient catalysis? Explain your answer.c) How would the pka for Lys change if it was near a negatively charged residue like Glu? Explain using equilibrium in partb.d) How would the pKa for Lys change if it was near a hydrophobic residue like Val? Explain using equilibrium in part b.

β-oxidation of fatty acid occurs in mitochondria. Before β- oxidation activation of fatty acid occurs in the cytosol with ATP and CoA. Fatty acyl CoA cannot cross the inner mitochondrial membrane because the inner membrane of mitochondria is impermeable to most acyl-CoA molecules. So, Carnitine-mediated transfer fatty acyl -CoA occurs into the mitochondrial matrix. Once fatty acyl-CoA is oxidized with oxidation of the β-carbon and long-chain fatty-acyl CoA inside mitochondrial matrix they are subjected to a repeated four-step process. Which removes two carbons from the chain successively until the last two carbon fragment is obtained. series of four reactions has yielded a fatty acyl-CoA shortened by two carbons and one molecule of acetyl-CoA. The formation of ketone bodies occurs in liver mitochondria when acetyl CoA is stored in large amounts more than required for fatty acid biosynthesis and oxidation. The process of formation of ketone bodies is known as ketogenesis. Ketone bodies include acetoacetate,β-hydroxybutyrate, and acetone. In acidosis, the level of acetoacetate, and β-hydroxybutyrate increase in the blood which leads to a lower blood pH creates an acidic environment. To generate energy ketone bodies are used by cardiac and skeletal tissues. In starvation conditions i.e., low amount of glucose brain also uses ketone bodies as a source of energy. Failing of heart is an energy-starved condition. In such a condition heart loses or reduces its ability to utilize fatty acids as fuel.Biosynthesis of ketone bodies in mammals is shown in the below figure Acetoacetate decarboxylase consists of antiparallel β-sheets and cone-shaped β -barrels. the active site in each protomer of enzyme surrounded by a core of cone-shaped β-barrel. Mostly hydrophobic amino acid residue present at the active site such as Tyr113,phe 27, and Met 97. The active site also contains two charged residues Arg 29 and Glu 76 which play important role in substrate binding and orienting the active site for substrate binding respectively. Hydrophobic environment favors neutral amine form of Lys115 that plays an important role in Schiff base formation. Schiff base intermediate form covalent bond with Lys 115 in the active site. Acetoacetate decarboxylase catalyzes the reaction decarboxylation producing acetone and co2 which is proceeded by forming Schiff base intermediate detail mechanism of action of acetoacetate decarboxylase shown in the following figure.