BIOQUÍMICA 2023

Nature and Classification of Enzymes

Enzymes are biological polymers that catalyze the chemical reactions essential for life as we know it. Their primary functions include the decomposition of nutrients to supply energy and chemical components. Structurally, enzymes are predominantly composed of proteins, with specific exceptions such as ribosomal RNA involved in protein synthesis and ribozymes, which facilitate the synthesis of ribosomal RNA. The fundamental enzymatic system consists of a Substrate, an Enzyme, and a resulting Product.

Related components include coenzymes, which act as shuttles transporting solutes or chemical groups between points in the cell while stabilizing molecules like $NADH$ and $FADH$. An example is Coenzyme A, which is critical for recognition handling. A prosthetic group is characterized by a tight, stable incorporation into the enzyme structure via covalent forces; examples include metalloenzymes, biotin, and Flavin Mononucleotide ($FMN$). Cofactors are similar to prosthetic groups but exhibit weak, transient, and dissociable binding, requiring proximity to the enzyme for the reaction to occur, as seen with $ATP$.

Enzymes are classified into six major categories based on the reactions they catalyze. Oxidoreductases catalyze oxidation and reduction reactions, such as the reduction of pyruvate to lactate by lactate dehydrogenase. Transferases catalyze the transfer of chemical groups, such as the hexokinase/glucokinase reaction that adds a phosphate group to glucose, forming glucose 6-phosphate. Hydrolases break chemical bonds using water ($H_2O$), exemplified by glucose 6-phosphatase converting glucose 6-phosphate back into glucose. Lyases break or generate double bonds, such as aldolase dividing fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Isomerases catalyze geometric or structural rearrangements, such as phosphotriose isomerase converting dihydroxyacetone phosphate to glyceraldehyde 3-phosphate. Finally, Ligases catalyze the union of two molecules in reactions coupled to $ATP$ hydrolysis, such as pyruvate carboxylase converting pyruvate and $CO_2$ into oxaloacetate using $ATP$.

Catalysis mechanisms vary depending on the chemical environment. Catalysis by proximity occurs within a specific zone to facilitate the reaction. Catalysis by strain involves forcing bonds to break through intense pressure. Covalent catalysis involves the temporary formation of a covalent bond that returns to its initial state. Acid-base catalysis occurs at the same $pH$ and is commonly observed in bacteria, though it is less frequent in the human body.

Vitamins and Their Coenzymatic Roles

Vitamins are organic nutrients required in small quantities for various biological functions. Because the body cannot synthesize them, they must be supplied through the diet. Vitamin $B_1$ (Thiamine) acts as a coenzyme or prosthetic group for enzymes such as pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and transcetolase. Vitamin $B_3$ (Niacin) is integral to dehydrogenases and forms $NAD^+$ and $NADP^+$ for oxidoreduction reactions. Vitamin $B_6$ (Pyridoxine) serves as a coenzyme in transamination and for glycogen phosphorylase during glycogenolysis. Vitamin $B_2$ (Riboflavin) is part of dehydrogenases and constitutes $FAD$ and $FMN$ for oxidoreductions. Vitamin $B_5$ (Pantothenic Acid) is a functional part of Coenzyme A ($CoA$) and is involved in gluconeogenesis and fatty acid synthesis. Vitamin $B_7$, $B_8$, or $H$ (Biotin) is essential for carboxylation in gluconeogenesis and fatty acid metabolism.

Isoenzymes are enzymes that perform the same function but are located in different organs or tissues. Glucokinase is primarily found in the liver and pancreas and is active during feeding, whereas Hexokinase is found in all cells and is active during fasting. Creatine Phosphokinase ($CK$) stores energy; its isoforms include $CK-MM$ (muscle), $CK-BB$ (brain), and $CK-MB$ (heart). Lactate Dehydrogenase ($LDH$) has five tetrameric combinations of Heart ($H$) and Muscle ($M$) subunits: $I_1$ ($HHHH$) in heart/erythrocytes, $I_2$ ($HHHM$) in heart/leukocytes, $I_3$ ($HHMM$) in lungs, $I_4$ ($HMMM$) in kidneys/pancreas/leukocytes, and $I_5$ ($MMMM$) in liver/skeletal muscle. Energy molecules also vary by organ; while $ATP$ is used by most organs, $GTP$ is preferred by gluconeogenic organs like the liver, kidney, and intestine.

Glycolysis: Aerobic and Anaerobic Pathways

Glycolysis is the principal pathway for the metabolism of glucose, fructose, galactose, and other carbohydrates, occurring in the cytosol of all cells. In aerobic conditions, pyruvate enters the mitochondria, undergoes oxidative decarboxylation to acetyl-CoA, and is oxidized to $CO_2$ in the Citric Acid Cycle. The aerobic sequence involves ten steps: 1) Glucose to Glucose 6-phosphate (via hexokinase/glucokinase, losing one $ATP$); 2) Glucose 6-phosphate to Fructose 6-phosphate (via phosphate hexokinase isomerase); 3) Fructose 6-phosphate to Fructose 1,6-bisphosphate (via phosphofructokinase, losing a second $ATP$); 4) Fructose 1,6-bisphosphate splits into Glyceraldehyde 3-phosphate and Dihydroxyacetone phosphate (via aldolase); 5) Dihydroxyacetone phosphate converts to Glyceraldehyde 3-phosphate (via phosphotriose isomerase); 6) Glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate (via glyceraldehyde 3-phosphate dehydrogenase, yielding $NADH+H^+$); 7) 1,3-bisphosphoglycerate to 3-phosphoglycerate (via phosphoglycerate kinase, gaining two $ATP$); 8) 3-phosphoglycerate to 2-phosphoglycerate (via phosphoglycerate mutase); 9) 2-phosphoglycerate to Phosphoenolpyruvate (via enolase, releasing $H_2O$ and requiring $Mg^{2+}$); and 10) Phosphoenolpyruvate to Pyruvate (via pyruvate kinase, gaining two more $ATP$).

Anaerobic glycolysis occurs when $NADH$ cannot be reoxidized by the respiratory chain due to a lack of oxygen. In several tissues, such as erythrocytes (which lack mitochondria), $NADH$ reduces pyruvate to lactate, catalyzed by lactate dehydrogenase, yielding $NAD^+$ to restart step 6 of glycolysis. The Rapoport-Luebering pathway is a bypass in erythrocytes where 1,3-bisphosphoglycerate is converted to 2,3-bisphosphoglycerate by bisphosphoglycerate mutase, then to 3-phosphoglycerate by 2,3-bisphosphoglycerate phosphatase; this pathway generates no $ATP$ but regulates oxygen release from hemoglobin.

Energy balance for Glycolysis: The net gain of $ATP$ per mole of glucose in anaerobic conditions is $2$ ($4$ gained minus $2$ lost). In aerobic conditions, including the oxidation of $2$ $NADH$ (which equals $2 \times 2.5 = 5$ $ATP$), the total yield is $7$ $ATP$.

Pyruvate Dehydrogenase Complex and The Krebs Cycle

Pyruvate formed in the cytosol is transported into the mitochondria via a proton or pyruvate symporter. The Pyruvate Dehydrogenase Complex ($PDH$) performs oxidative decarboxylation to form acetyl-CoA. This complex requires thiamine diphosphate ($TDP$), lipoamide, $NAD^+$, $FAD$, and $CoA$. The process involves three steps: 1) Pyruvate decarboxylation by pyruvate dehydrogenase and reaction with oxidized lipoamide to form acetyl lipoamide; 2) Acetyl lipoamide reacts with $CoA$ to form acetyl-CoA and reduced lipoamide; 3) Reduced lipoamide is reoxidized by dihydrolipoyl dehydrogenase using $FAD$, which then transfers electrons to $NAD^+$ for the respiratory chain. The reaction is: $Pyruvate + NAD^+ + CoA \rightarrow Acetyl-CoA + NADH + H^+ + CO_2$. This generates $2.5$ $ATP$ per pyruvate ($5$ per glucose). Thiamine deficiency leads to lactic and pyruvic acidosis.

The Citric Acid Cycle (Krebs Cycle) occurs in the mitochondria as the final common pathway for the oxidation of all macronutrients. The steps are: 1) Acetyl-CoA condenses with oxaloacetate to form citrate (via citrate synthase); 2) Citrate isomerizes to isocitrate via cis-aconitate (via aconitase); 3) Isocitrate is oxidized to oxalosuccinate (via isocitrate dehydrogenase); 4) Oxalosuccinate decarboxylates to alpha-ketoglutarate (requiring $Mg^{2+}$ or $Mn^{2+}$); 5) Alpha-ketoglutarate undergoes oxidative decarboxylation to succinyl-CoA (via a multi-enzyme complex requiring $TDP$, lipoate, $NAD^+$, $FAD$, and $CoA$); 6) Succinyl-CoA to succinate (via succinate thiokinase, generating $GTP/ATP$); 7) Succinate to fumarate (via succinate dehydrogenase, reducing $FAD$ to $FADH_2$); 8) Fumarate hydrates to malate (via fumarase); 9) Malate is oxidized to oxaloacetate (via malate dehydrogenase). Inhibitors include fluoroacetate (aconitase), malonate (succinate dehydrogenase), and Arsenite (alpha-ketoglutarate dehydrogenase).

Electron Transport Chain and Chemical Shuttles

The Electron Transport Chain ($ETC$) captures energy released during oxidation. Complex I ($NADH$-Q oxidoreductase) transfers electrons from $NADH$ to Coenzyme Q ($Ubiquinone$). Complex II ($Succinate$-Q reductase) transfers electrons from $FADH_2$ to Q. Complex III (Q-cytochrome c oxidoreductase) passes electrons to cytochrome c. Complex IV (cytochrome c oxidase) transfers electrons to oxygen. Chemiosmosis involves the $ATP$ synthase using the proton gradient to convert $ADP + Pi$ into $ATP$. Per complex, $1$ $NADH$ yields $2.5$ $ATP$ and $1$ $FADH_2$ yields $1.5$ $ATP$. The Q cycle is necessary because cytochromes carry one electron while ubiquinone carries two.

The Malate-Aspartate Shuttle operates in the heart, liver, and kidney to transport reducing equivalents into the mitochondria. Cytosolic malate dehydrogenase reduces oxaloacetate to malate using $NADH$. Malate enters the mitochondria and is reoxidized to oxaloacetate, producing mitochondrial $NADH$ ($2.5$ $ATP$). Oxaloacetate then undergoes transamination with glutamate to form aspartate and alpha-ketoglutarate, which exit back to the cytosol to reset the cycle.

Gluconeogenesis, Glycogen Metabolism, and Alternative Pathways

Gluconeogenesis is the synthesis of glucose from non-carbohydrate substrates, including glucogenic amino acids (like alanine), lactate, glycerol, and propionate. Glycerol is phosphorylated by glycerol kinase to glycerol 3-phosphate and سپس oxidized to dihydroxyacetone phosphate. Lactate follows the Cori Cycle from muscle/erythrocytes to the liver to be converted into pyruvate. Propionate converts to succinyl-CoA. Alanine follows the Cahill Cycle. To bypass irreversible glycolytic steps, gluconeogenesis uses: 1) Glucose 6-phosphatase (bypasses hexokinase); 2) Fructose 1,6-bisphosphatase (bypasses phosphofructokinase); and 3) Pyruvate carboxylase and phosphoenolpyruvate carboxykinase (bypasses pyruvate kinase).

Glycogenesis is the formation of glycogen for storage, primarily in the liver and muscle. It starts with glucose 6-phosphate converting to glucose 1-phosphate (phosphoglucomutase), then to $UDPGlc$ ($UDPGlc$ pyrophosphorylase). Glycogenin catalyzes the initial seven glucose residues to form a primer for glycogen synthase ($1 \rightarrow 4$ bonds). The branching enzyme creates $1 \rightarrow 6$ bonds every $11$ residues. Glycogenolysis is the breakdown of glycogen during fasting, initiated by glycogen phosphorylase which produces glucose 1-phosphate. Glucano transferase and debranching enzymes handle the branches. Regulation is controlled by $cAMP$; glucagon and epinephrine increase $cAMP$ to activate phosphorylase, while insulin antagonizes this.

The Pentose Phosphate Pathway provides $NADPH$ for fatty acid/steroid synthesis and ribose 5-phosphate for nucleotides. It has an irreversible oxidative phase (Glucose 6-P to Ribulose 5-P yielding $2$ $NADPH$) and a reversible non-oxidative phase (transketolase and transaldolase reordering sugars). The Uronic Acid Pathway converts glucose to glucuronic acid for conjugation and excretion of metabolites/drugs.

Lipid Metabolism and Transport

Lipids are transported in the blood as lipoproteins. Apolipoproteins serve structural roles ($Apo-48$, $Apo-B100$), act as cofactors ($Apo \text{ C-II}$), or ligands ($Apo \text{ B-100}$, $Apo \text{ E}$). Chylomicrons transport dietary triacylglycerols from the intestine; once in circulation, they acquire $Apo \text{ C}$ and $Apo \text{ E}$ from $HDL$. Lipoprotein lipase ($LPL$) on capillary walls hydrolyzes them into free fatty acids and glycerol. Residual chylomicron remnants are taken up by the liver via receptor-mediated endocytosis. $VLDL$ carries lipids from the liver and is metabolized into $IDL$ and then $LDL$. $LDL$ particles, rich in cholesterol, are taken up by liver ($70$%) and extrahepatic tissues ($30$%). High plasma $LDL$ is associated with atherosclerosis.

Lipogenesis occurs in the cytosol of the liver, kidney, lung, adipose tissue, and lactating mammary glands. Acetyl-CoA is transported from mitochondria to cytosol as citrate. The rate-limiting step is catalyzed by Acetyl-CoA carboxylase, which converts acetyl-CoA to malonyl-CoA using biotin and $ATP$. Then, the fatty acid synthase complex (a homodimer with six enzymatic activities) builds palmitate.

Fatty acid oxidation ($\beta$-oxidation) occurs in the mitochondria. Long-chain fatty acids are activated to Acyl-CoA and transported via the Carnitine shuttle. The four