Enzyme Kinetics P1

Elevated levels of homocysteine may be detectable in urine, and a cyanide-nitroprusside test can screen for sulfur-containing amino acids like homocystine, turning the urine red-purple if positive.

BCKDH is involved in the catabolism of branched-chain amino acids (BCAAs) by decarboxylating their corresponding a-keto acids into acyl-CoA derivatives for energy production.

Biochemistry plays a central role in life sciences by providing insights into the molecular mechanisms of biological processes. It is crucial for understanding disease mechanisms, developing diagnostic tools, and creating therapeutics in medicine.

• pH: A measure of the acidity or basicity of a solution.
• Ka: The acid dissociation constant, indicating the strength of an acid in solution.
• pKa: The negative logarithm of Ka, providing a more convenient way to express acid strength.

These concepts are important for understanding enzyme activity, metabolic pathways, and buffer systems in biological systems.

The Henderson-Hasselbalch equation is used to calculate the pH of a buffer solution based on the concentration of the acid and its conjugate base. It is expressed as:

pH = pKa + log([A-]/[HA])

This equation helps in understanding how changes in concentrations affect the pH, which is critical for enzyme function and metabolic reactions.

Gibbs free energy (ΔG) indicates the spontaneity of a reaction.

• If ΔG < 0, the reaction is spontaneous.
• If ΔG > 0, the reaction is non-spontaneous.

Understanding Gibbs free energy is essential for predicting the direction of biochemical reactions and their equilibrium states.

• Acid: A substance that donates protons (H+).
• Base: A substance that accepts protons.
• Conjugate Acid: The species formed when a base gains a proton.
• Conjugate Base: The species that remains after an acid donates a proton.

These definitions are crucial for understanding buffer systems and enzyme catalysis in biological systems.

• Water: Acts as a solvent for biochemical reactions, participates in hydrolysis, and helps maintain cellular structure.
• Buffers: Maintain stable pH levels in biological systems, which is vital for enzyme activity and metabolic processes.

Both are essential for maintaining homeostasis in living organisms.

ATP (adenosine triphosphate) is the primary energy currency of the cell. It is involved in:

1. Phosphoryl group transfer: ATP donates a phosphate group to other molecules, facilitating energy transfer.
2. Driving endergonic reactions: ATP hydrolysis provides the energy needed for various biochemical processes, including muscle contraction and active transport.

Thus, ATP is crucial for energy metabolism.

Amino acids can be classified based on:

This classification is important for understanding protein structure and function.

The isoelectric point (pI) is the pH at which an amino acid has no net charge. At this point, the amino acid is least soluble and can precipitate out of solution. Understanding pI is important for techniques like isoelectric focusing and for predicting the behavior of proteins in different pH environments.

Clinical correlations include:

• Alkaptonuria: A metabolic disorder affecting tyrosine metabolism.
• Homocystinuria: A disorder of methionine metabolism.
• Maple Syrup Urine Disease: Affects branched-chain amino acid metabolism.
• Phenylketonuria: A disorder of phenylalanine metabolism.
• Respiratory and metabolic acidosis/alkalosis: Conditions affecting acid-base balance.

These disorders highlight the importance of understanding biochemical pathways in diagnosis and treatment.

Biochemistry is the science concerned with the molecules present in living organisms, individual chemical reactions and their enzyme catalysts, and the expression and regulation of each metabolic process or pathway.

Important functional groups in biochemical reactions include:

1. Alcohols
2. Aldehydes
3. Ketones
4. Carboxyl groups
5. Anhydrides
6. Sulfhydryl groups
7. Amines
8. Esters
9. Amides

Biochemistry, medicine, and other health care disciplines are intimately related. Health depends on a harmonious balance of biochemical reactions, while disease reflects abnormalities in biomolecules, biochemical reactions, or processes. Biochemical approaches help illuminate disease causes and design therapies, and biochemical tests are integral for diagnosis and treatment monitoring.

Genetic mutations in enzyme formation can cause the accumulation of starting materials, leading to amino acid metabolic disorders such as phenylketonuria, maple syrup urine disease, homocystinuria, and alkaptonuria.

The acidic amino acids with negative charges on their side chains at pH 7 are aspartate and glutamate.

Biochemistry is foundational for several disciplines including:

1. Molecular biology
2. Cell biology
3. Genetics
4. Nutrition
5. Organic chemistry
6. Computational biology
7. Spectroscopy
8. Biophysics
9. Developmental biology
10. Microbiology
11. Physiology
12. Pathology
13. Pharmacology
14. Immunology

A biochemical reaction is the transformation of one molecule into a different molecule inside a cell, mediated by enzymes, which are biological catalysts that alter the rate and specificity of chemical reactions.

Biochemical reactions are classified according to the functional groups that react, such as hydroxylation, decarboxylation, and methylation.

Hydroxylation is a chemical process that introduces a hydroxyl group (-OH) into an organic compound, often facilitated by enzymes called hydroxylases.

Decarboxylation is a chemical reaction that removes a carboxyl group and releases CO2.

In oxidation reactions, electrons are lost, while in reduction reactions, electrons are gained.

Water is a dipolar molecule that forms hydrogen bonds, acts as a solvent, absorbs energy, and buffers living systems from chemical changes necessary to sustain life.

The body maintains fluid homeostasis through the intake of water influenced by availability, thirst, and hunger, and adjusts urinary excretion to compensate for variations in water loss and intake, regulated by hormones like aldosterone and antidiuretic hormone (ADH).

pH is an inverse measure of the hydrogen ion concentration of a solution. It is calculated using the formula:
pH = -log [H+]. Lower pH values indicate a more acidic solution.

Strong acids completely dissociate in water, releasing all their hydrogen ions, while weak acids only partially dissociate. The strength of a weak acid is quantified by its dissociation constant (Ka); a larger Ka indicates a stronger acid.

The relationships are defined as follows:

• pOH = -log [OH]
• pKa = -log Ka
• pH + pOH = 14. This means that knowing any one of these values allows you to calculate the others.

The pH scale categorizes solutions as follows:

• Acidic (pH < 7): Examples include 1 M HCl, gastric juice, lemon juice, and cola.
• Neutral (pH = 7): Examples include milk, human blood, and seawater.
• Basic (pH > 7): Examples include baking soda solution, household ammonia, and bleach.

A conjugate base is formed after an acid loses its proton (H+).

A conjugate acid is formed after a base gains a proton (H+).

Buffers resist changes in pH by maintaining a mixture of undissociated acid and its conjugate base, allowing the solution to resist changes when H+ or OH- is added.

The effectiveness of a buffer is determined by:

1. Its pKa relative to the pH of the solution.
2. Its concentration.

The normal pH range for blood is 7.35 to 7.45, maintained primarily by the carbonic acid/bicarbonate (H2CO3/HCO3) buffer system.

When blood pH is lower than normal, it leads to acidosis; when higher, it leads to alkalosis. These conditions can be caused by hypoventilation, hyperventilation, and metabolic disturbances.

pKa is an inverse measure of the tendency for a molecule to donate protons (act as an acid); the smaller the pKa value, the stronger the acid.

The Henderson-Hasselbalch equation relates the pH of a solution to the Ka of an acid and the extent of its dissociation, allowing for the calculation of pH based on the ratio of conjugate base to acid.

When the ratio [A-]/[HA] is 100:1, the pH is calculated as:

pH = pKa + log(100) = pKa + 2.

When an acid is exactly half-neutralized, [A-] = [HA], and the pH is:

pH = pKa + log(1) = pKa + 0, thus pH = pKa.

When the ratio [A-]/[HA] is 1:10, the pH is calculated as:

pH = pKa + log(1/10) = pKa - 1.

The major source of acid in the body is CO2, which reacts with water to form carbonic acid. This acid dissociates to produce bicarbonate and a proton, influencing pH levels. The body regulates pH through respiratory mechanisms that remove carbonic acid by expiring CO2 and through renal excretion of acids as ammonium (NH4+) and other ions.

Carbonic anhydrase is an enzyme found in erythrocytes that facilitates the conversion of carbon dioxide from tissues into bicarbonate in the blood. It also aids in the breakdown of bicarbonate to release carbon dioxide in the lungs, ensuring efficient gas exchange necessary for health.

The lungs maintain body pH by expiring CO2, which helps regulate the levels of carbonic acid in the blood. The kidneys contribute by excreting acids, primarily in the form of ammonium (NH4+), which helps to eliminate excess protons and maintain a neutral pH.

The relationship is described by the equation pH = pKa + log([HCO3-]/[CO2]). This indicates that pH is directly proportional to bicarbonate concentration and inversely proportional to the partial pressure of carbon dioxide (pCO2).

The primary buffers in body fluids include:

1. Bicarbonate
2. Phosphate
3. Hemoglobin

These buffers work to neutralize excess acids and maintain a stable pH in the body.

Respiratory acidosis is caused by hypoventilation, leading to elevated levels of blood CO2. This increase in CO2 results in a higher concentration of [H+] in the blood, which decreases the pH.

Metabolic acidosis occurs when a strong acid is added to the blood or when bicarbonate (HCO3) is lost, leading to a decrease in blood pH.

Hyperventilation causes a decrease in blood CO2 levels, which lowers the [H+] concentration in the blood, resulting in an increased pH and leading to respiratory alkalosis.

Metabolic alkalosis can be caused by the ingestion of a strong base or the loss of acids, resulting in an increase in blood pH.

The relationship is described by the equation: pH = pKa + log [conjugate base]/[acid]. A decrease in pH corresponds to an increase in [H+], while an increase in pH corresponds to a decrease in [H+].

In response to respiratory acidosis, the body increases renal retention of HCO3 (more base) to buffer the excess acid from CO2 retention.

During metabolic acidosis, there is more acid generated, leading to hypoventilation and less CO2 being released in breath. The HCO3 levels will be low as the body attempts to buffer the acid.

In metabolic alkalosis, the body compensates by increasing the release of CO2 through hyperventilation and renal excretion of HCO3, which helps to lower the pH back towards normal.

During anaerobic exercise, lactic acid is produced from glucose through anaerobic glycolysis. Lactic acid dissociates into lactate and protons (H+). The bicarbonate buffer system responds by reacting bicarbonate (HCO3-) with protons to form carbonic acid (H2CO3), which then breaks down into water (H2O) and carbon dioxide (CO2). This increase in CO2 stimulates hyperventilation to expel the excess CO2 from the body.

Gibbs free energy represents the work available from chemical reactions, which can be harnessed to perform functions of living organisms. It is determined by the equation ΔG = ΔH - TΔS, where ΔH is the change in enthalpy, T is the temperature, and ΔS is the change in entropy.

A negative ΔH indicates heat flow out of a system, leading to more stable, lower energy products from reactants.

The equilibrium constant (Keq) is a ratio calculated from the concentrations of reactants and products at equilibrium, expressed as Keq = ([X]^x[Y]^y)/([A]^a[B]^b) for the reaction aA + bB ⇄ xX + yY.

A positive ΔG° indicates that the reaction is non-spontaneous under standard-state conditions, favoring the reactants at equilibrium.

Le Chatelier's Principle states that systems at equilibrium will respond to applied stress by shifting to reduce that stress. Increasing reactant concentration drives the reaction forward to produce more product, thereby correcting the concentration stress.

Endothermic reactions require the transfer of heat into the system (+ΔH), while exothermic reactions transfer heat out of the system (-ΔH).

A reaction will spontaneously occur if ΔG < 0 (exergonic reaction).

ATP hydrolysis (ΔG << 0) is thermodynamically favorable and allows ATP to act as an energy currency by coupling its hydrolysis with thermodynamically unfavorable reactions, enhancing overall favorability.

Depending on the nucleophilic attack on the α, β, or γ phosphate of ATP, different group transfers can occur: a single phosphoryl group, a pyrophosphoryl group, or an adenylyl group (AMP with inorganic pyrophosphate released).

ATP is involved in energy production from carbohydrates, lipids, and proteins, and in energy utilization for muscle contraction, active ion transport, biosynthesis, detoxification, and thermogenesis.

The nine essential amino acids are:

1. Histidine
2. Isoleucine
3. Leucine
4. Lysine
5. Methionine
6. Phenylalanine
7. Threonine
8. Tryptophan
9. Valine

Hydrophobic amino acids have side chains that contain either aliphatic groups (e.g., valine, leucine, isoleucine) or aromatic groups (e.g., phenylalanine, tyrosine, tryptophan) that can form hydrophobic interactions. Tyrosine, however, has a phenolic group that can carry a negative charge above its pKa (~10.5), making it less hydrophobic in that pH range.

Ionizable groups present on the side chains of seven amino acids can carry a charge depending on the pH. When charged, they can form electrostatic interactions, which are crucial for protein structure and function.

Asparagine and glutamine have amide groups present in their side chains, which distinguishes them from other amino acids that may have different functional groups.

Below its pKa (3.9), aspartate predominates in the form CH₂-COOH. Above its pKa, it predominates in the form of negatively charged COO⁻ + H⁺.

Disulfide bonds, formed by the oxidation of sulfhydryl groups in cysteine residues, play a critical role in stabilizing the three-dimensional structure of proteins.

At physiologic pH (~7.4), the α-amino group is protonated and carries a positive charge, while the α-carboxyl group is deprotonated and carries a negative charge.

The basic amino acids with positive charges on their side chains at pH 7 are arginine, lysine, and histidine.

The isoelectric point (pI) is the pH at which the number of positive charges equals the number of negative charges, resulting in an overall charge of zero for the amino acid.

As the pH of the solution increases, the protons on the amino acids dissociate. At low pH, amino acids keep their protons, but as the pH rises above the pKa of an ionizable group, that group becomes vulnerable to deprotonation.

For an amino acid without an ionizable side chain, two pKa values are observed:

1. pK₁ corresponds to the a-carboxyl group (approximately 2).
2. pK₂ corresponds to the a-amino group (approximately 9).

For an amino acid with an ionizable side chain, three pKa values are observed:

1. pK₁ for the a-carboxyl group (about 2).
2. pK₂ for the a-amino group (about 9).
3. A third pKa that varies with the amino acid and depends on the pKa of the side chain.

The isoelectric point (pI) is the pH at which a protein has no net electric charge. It is influenced by the anionic or cationic character of the protein's amino acid side chains at a certain pH.

At the pKa value of the carboxyl group of glycine (2.34), the amino acid exists as two species: 50% with a protonated carboxyl group and 50% with a deprotonated carboxyl group, indicating a balance between the two forms.

When the pH of the solution moves 2 units above the pKa value of the carboxyl group (around pH 4.34), the deprotonated form of the carboxyl group becomes almost 100%, indicating that the group is fully deprotonated under these conditions.

Cysteine = C, Histidine = H, Isoleucine = I, Methionine = M, Serine = S, Valine = V

Alanine = A, Glycine = G, Leucine = L, Proline = P, Threonine = T

Arginine = R, Asparagine = N, Aspartate = D, Glutamate = E, Glutamine = Q, Phenylalanine = F, Tyrosine = Y, Tryptophan = W

Aspartate or Asparagine = B, Glutamate or Glutamine = Z, Lysine = K, Undetermined amino acid = X

Amino acids are composed of:

1. α-Carboxyl group (-COOH)
2. α-Amino group (-NH2)
3. Side chains (R groups) (20 different ones)

At physiologic pH, the α-Carboxyl group is deprotonated and exists as COO-.

At physiologic pH, the α-Amino group is protonated and exists as NH3+.

When protonated, side chains can release protons (H+) and act as weak acids, contributing to the buffering capacity of proteins.

The Henderson-Hasselbalch equation predicts the buffering capacity of amino acids, indicating how they can resist changes in pH.

The nonpolar side chains include:

• Alanine
• Glycine
• Isoleucine
• Leucine
• Methionine
• Phenylalanine
• Proline
• Tryptophan
• Valine

Acidic side chains, such as Aspartic acid and Glutamic acid, are characterized by a side chain that dissociates to COO- at physiologic pH.

Basic side chains, including Arginine, Histidine, and Lysine, are characterized by a side chain that is protonated and generally has a positive charge at physiologic pH.

In proteins, most α-COO- and α-NH3+ groups of amino acids are combined through peptide bonds, making them unavailable for chemical reactions.

The chemical nature of the side chain determines the role that the amino acid plays in a protein, particularly in how the protein folds into its native conformation.

Deficiency in Homogentisate Oxidase leads to alkaptonuria, characterized by dark urine, ochronosis (pigment accumulation in tissues), and joint issues due to the buildup of homogentisic acid.

A deficiency in Cystathionine Synthase results in homocystinuria, which is associated with intellectual disability, lens dislocation, and an increased risk of thromboembolism.

Deficiency in Branched-Chain α-Keto Acid Dehydrogenase leads to maple syrup urine disease (MSUD), characterized by neurotoxic keto acid accumulation, developmental delay, and sweet-smelling urine.

A deficiency in Phenylalanine Hydroxylase causes phenylketonuria (PKU), which can lead to intellectual disability and seizures if untreated, due to the accumulation of phenylalanine.

Homogentisate oxidase catalyzes the conversion of homogentisic acid into maleylacetoacetate, facilitating the breakdown of tyrosine and phenylalanine into energy and other metabolites.

A deficiency in homogentisate oxidase causes alkaptonuria, a rare autosomal recessive disorder characterized by the accumulation of homogentisic acid.

Urine may initially appear normal but darkens upon standing due to oxidation of homogentisic acid. A specific test using ferric chloride can confirm alkaptonuria by turning the urine purple-black.

Cystathionine synthase converts homocysteine and serine into cystathionine, which is critical for producing cysteine and regulating homocysteine levels.

Deficiency in cystathionine synthase leads to homocystinuria, an autosomal recessive disorder characterized by elevated homocysteine levels that can damage blood vessels and tissues.

Deficiency results in maple syrup urine disease (MSUD), a metabolic disorder characterized by the accumulation of branched-chain amino acids (BCAAs) and their keto acids, leading to neurological damage and a characteristic sweet odor in bodily fluids.

The urinalysis results for MSUD may include:

• Sweet, maple syrup-like odor due to the accumulation of branched-chain keto acids.
• Ketoaciduria with elevated organic acids like α-ketoisocaproate, α-keto-β-methylvalerate, and α-ketoisovalerate, detectable via organic acid analysis (e.g., gas chromatography-mass spectrometry).
• A ferric chloride test may yield a gray-green color, indicating the presence of keto acids.
• Routine urinalysis is typically normal unless secondary effects, such as dehydration, occur.

Comparing the amino acid profiles helps in identifying metabolic disorders. In MSUD, there is an abnormal accumulation of certain amino acids, which can be detected through mass spectrometry. This comparison aids in early diagnosis and management of the condition.

In the MSUD amino acid profile, amino acids such as Leucine, Isoleucine, and Valine are typically elevated compared to a normal profile, indicating a metabolic disruption.

The mass spectrometry results show that normal patients have a balanced amino acid profile, while MSUD patients exhibit elevated levels of specific branched-chain amino acids, particularly Leucine, Isoleucine, and Valine, indicating a metabolic disorder.

Phenylalanine hydroxylase (PAH) converts phenylalanine into tyrosine in the liver, requiring tetrahydrobiopterin (BH4) as a cofactor. This is the first step in phenylalanine metabolism.

A deficiency of phenylalanine hydroxylase causes phenylketonuria (PKU), an autosomal recessive disorder characterized by the accumulation of phenylalanine, leading to intellectual disability and other neurological issues if untreated.

Key urinalysis findings for PKU include elevated phenylalanine and its metabolites, a green color change in the ferric chloride test due to phenylpyruvic acid, and a 'mousy' or musty odor due to phenylacetic acid.

The ferric chloride test turns urine green due to the presence of phenylpyruvic acid, which is a classic screening method for phenylketonuria (PKU).

Urine in patients with phenylketonuria (PKU) may have a 'mousy' or musty odor due to the presence of phenylacetic acid.

Water is distributed between intracellular and extracellular compartments, with the extracellular compartment comprising interstitial fluids, blood, and lymph.

The pH of a solution is the negative log of its hydrogen ion concentration. A lower pH indicates a higher concentration of hydrogen ions, making the solution more acidic.

A strong acid dissociates almost completely in water to release hydrogen ions, while a weak acid only partially dissociates, characterized by its dissociation constant (Ka).

A buffer is a mixture of undissociated acid and its conjugate base that resists changes in pH when H⁺ or OH⁻ is added. Its effectiveness is greatest near its pKa.

The isoelectric point (pI) is the pH at which the number of positive charges equals the number of negative charges on an amino acid, resulting in an overall charge of zero.

Gibbs free energy represents the work available from chemical reactions and is calculated using the equation ΔG = ΔH - TAS.

Carbon dioxide reacts with water to form carbonic acid, which dissociates to produce bicarbonate and protons, playing a crucial role in maintaining pH balance in body fluids.

Amino acid metabolism — specifically phenylalanine metabolism due to phenylalanine hydroxylase deficiency.

The hydrolysis of ATP into ADP + P₁ is a thermodynamically favorable reaction (ΔG << 0), allowing ATP to act as an energy currency by coupling with unfavorable reactions to drive them forward.

Repeated vomiting of stomach contents, including HCl

The total amount of carbonic anhydrase in red blood cells is decreased in anemia, reducing bicarbonate buffer capacity.

Coupling allows the energetically unfavorable phosphorylation of glucose to proceed by using the energy from ATP hydrolysis.

Almost completely unprotonated (~100% -COO-).

Amino acids — specifically impaired metabolism of the amino acid phenylalanine (phenylalanine hydroxylase deficiency).

Repeated vomiting of stomach contents, including HCl — loss of acid leads to metabolic alkalosis.

Anemia reduces the number of red blood cells and thus the total carbonic anhydrase available to catalyze H2CO3 ⇌ CO2 + H2O, decreasing buffer capacity.

Coupling makes the net process energetically favorable, allowing phosphorylation of glucose to proceed via energy from ATP hydrolysis.

Almost completely deprotonated (≈100% -COO-).