Enzyme Regulation and Clinical Enzymology

Quantitative Assay of Enzymes

• Enzyme activity is measured by the rate at which a substrate is transformed into a product.
• The greater the transformation rate, the higher the enzyme activity.
• Enzyme activity can be estimated from the rate of product appearance or substrate disappearance.

Measurement of Enzyme Activity

• One unit of enzyme activity is the amount causing the transformation of 1.0 mmol of substrate per minute at 25°C under optimal conditions.
• Enzyme activity is usually expressed as mmol of substrate disappeared or mmol of product formed per minute.

Turnover Number of Enzymes

• Turnover number refers to the number of substrate molecules a single enzyme molecule transforms in a specific unit of time when enzyme concentration is the rate limiting factor.
• Carbonic anhydrase has a high turnover number of 36,000,000.
• Examples of specific enzyme turnover numbers are listed for carbonic anhydrase, B-amylase, phosphoglucomutase, and fumarase.

Reaction Activation Energy

• All chemical reactions require energy to initiate the reaction, this is called activation energy.
• Enzymes reduce activation energy, allowing reactions to proceed more readily.
• An enzyme-catalyzed reaction needs less energy input than if the reaction had no enzyme.

The Activation Energy Barrier

• Chemical reactions involve breaking and forming bonds, requiring activation energy.
• Activation energy usually arises from thermal energy absorbed from surroundings.
• Enzymes catalyze reactions by lowering the activation energy barrier.
• Enzymes do not have an effect on the change in free energy (∆G).

Energy Changes during the Reaction

• All chemical reactions have an energy barrier between reactants and products, which is referred to as the free energy of activation.
• Free energy is a thermodynamic function useful in understanding enzyme-catalyzed reactions.
• The change in free energy, denoted as ∆G, is the difference in free energy between reactants and products.
• If ∆G is negative, the reaction is spontaneous.
• If ∆G is positive, the reaction needs energy input.

Exergonic and Endergonic Reactions

• Exergonic reactions release free energy; they are spontaneous.
• Endergonic reactions absorb free energy; they are not spontaneous.

ATP powers cellular work by coupling exergonic reactions to endergonic reactions

• Cells manage energy resources by energy coupling, using energy from one process to drive another.
• ATP is a common energy currency in cells for coupling exergonic and endergonic reactions.

Structure of ATP and its Hydrolysis

• ATP consists of an adenine, a ribose sugar, and three phosphate groups connected to each other by high-energy phosphate bonds.
• Hydrolysis of ATP releases energy from breaking the terminal phosphate bond.
• The energy comes from the chemical change to a state of lower free energy, not from the phosphate bonds themselves.

The Regeneration of ATP

• ATP is continuously regenerated from ADP through addition of a phosphate group.
• The energy for phosphorylating ADP comes from catabolic reactions within the cell.
• The ATP cycle efficiently shuttles energy between catabolism and anabolism.

Mechanisms for Regulating Enzyme Activity

• Enzymes' activity regulation is crucial for coordinating metabolic processes.
• There are three main mechanisms: allosteric modulation, covalent modification, and induction/repression of enzyme synthesis.

Allosteric Modulation

• Allosteric enzymes exist in different structural forms (high vs low).
• Allosteric modulators (ligands) bind to a site separate from the active site, affecting the enzyme activity.
• Binding may either activate or inhibit the enzyme.
• The allosteric site is distinct from the substrate-binding site.

Allosteric Modulators

• Allosteric modulators bind to the allosteric site by non-covalent interactions.
• These interactions reversibly alter the enzyme's conformation and its activity.

Allosteric Inactivators and Activators

• Allosteric activators increase enzyme activity, while inhibitors decrease it.
• Activator binding alters conformation to the high-affinity state.
• Inhibitor binding promotes low affinity active conformation.

Most Allosteric Enzymes

• Most are oligomeric, with multiple subunits (protomers).
• Binding of substrate to one subunit may affect binding affinity of other subunits in a positive or negative manner (cooperative).
• Some are monomeric enzymes.

Homotropic Effect and Heterotropic Effect

• Homotropic effect occurs when the substrate acts as an allosteric modulator, often leading to positive cooperativity.
• Heterotopic effect occurs when a different molecule (like an end product) acts as an allosteric modulator, often acting as feedback regulation of the enzyme activity.

Example of Feedback Inhibition

• Aspartate transcarbamoylase (ATCase) is an allosteric enzyme crucial for pyrimidine biosynthesis.
• CTP is an allosteric inhibitor for ATCase (heterotrophic effect) that effectively regulates the reaction.
• ATP, an intermediate in the pathway, acts as an allosteric activator (homotrophic effect) for ATCase.

Covalent Modification

• Enzyme activity is regulated by reversible covalent modifications.
• Key examples are modification by phosphorylation and dephosphorylation.
• Covalent modification alters the charge state of the enzyme's active site influencing its conformation and thus activity.

Example on Reversible Covalent Regulation

• Phosphorylation/dephosphorylation by protein kinases and phosphatases typically modify the active site for the enzyme in a reversible manner altering conformation. This results in altered activity.

Irreversible Activation by Proteolytic Cleavage

• Some proteins are produced in inactive zymogen forms.
• Activation occurs through proteolytic cleavage, a process where a protease cleaves a small portion from the protein.
• This triggers a conformational change enabling the zymogen to function in its active form.
• Example: activation of trypsin and pepsin from their respective zymogens, trypsinogen, and pepsinogen.

Induction-Repression of Enzyme Synthesis

• Enzyme synthesis can be controlled through gene expression.
• Enzyme synthesis can increase or decrease in response to cellular conditions (induction/repression).
• Changes in the synthesis rate affect intracellular enzyme concentration, hence modifying the enzyme's activity.

Examples of Induction and Repression

• Sex hormones, such as estrogen and testosterone, can induce or repress specific enzymes.
• Insulin can alter the synthesis rates of enzymes involved in glycolysis and gluconeogenesis.

Isoenzymes

• Related enzymes with similar metabolic functions.
• Isoenzymes may have different subunit compositions, thus different kinetics and/or regulatory behaviors and/or different tissue distributions.
• Different subunit compositions give them distinct properties, like different charge patterns, allowing separation by electrophoresis.

Mechanisms for Production of Isoenzymes

• Different combinations of polypeptide subunits create various isoenzyme forms.
• Example: creatine kinase (CK) is a dimer with muscle (M) and brain (B) subunits.
• Example: lactate dehydrogenase (LDH) is a tetramer with different subunit combinations.

Post-Translational Modification

• Post-translational modifications (PTMs) like adding phosphate, acetate, amide or methyl groups can change the properties of enzymes.
• This can lead to creation of isoenzymes by covalent addition.

Separation of Isoenzymes

• Isoenzyme separation is often achieved through electrophoresis based on the different properties and charges of the different isoenzymes, which are a product of different subunit composition.

Use of Inhibitors

• Isoenzyme activity can be inhibited by certain compounds.
• This can be helpful for identification.
• Example: Alkaline phosphatase (ALP) is inhibited by phenylalanine.

Enzymes in Clinical Diagnosis

• Enzyme assays in blood allow for diagnosis of various conditions.
• Cellular enzymes are usually localized to specific cells.
• During normal turnover, enzymes are released into plasma and then eliminated from the body.
• Under pathological conditions, enzyme levels can rise in plasma as a result of cell damage, which can indicate the severity of the pathology, and the specific region damaged.

Transaminases

• AST or GOT and ALT or GPT are important in diagnosing diseases of the hepatic, cardiac or skeletal-muscular systems.
• Higher levels of transaminases can indicate various conditions including acute pancreatitis.

Alkaline Phosphatases (ALP)

• ALP catalyzes the removal of phosphate groups from phosphate esters.
• Abundant in liver, bone, intestine and placenta.
• Elevated ALP levels can suggest bone or liver pathology

Acid Phosphatase (ACP)

• ACP hydrolyzes phosphate from phosphate esters at an acid pH.
• It's vital in diagnosing metastatic prostate cancers, since higher levels are associated with the presence and spread of such a cancer.

Lactate Dehydrogenase (LDH)

• LDH catalyzes the reversible interconversion of lactate and pyruvate.
• Found in most tissues, its elevated levels often indicate conditions like myocardial infarction or hemolytic anemia.
• Specific types of LDH, based on different subunit compositions, can be distinguished, thus contributing to the diagnostic value of assessing LDH serum levels.

Creatine Kinase (CK)

• CK catalyzes the transfer of phosphate to ADP from creatine phosphate, creating ATP.
• Abundant in heart and skeletal muscles, its raised levels often suggest conditions related to muscle damage.
• Different isoenzymes occur in different tissues (brain, heart, skeletal muscle) which aids in the diagnostic process, as the elevated levels are tissue specific.

Troponin T and Troponin I

• These regulatory proteins are involved in myocardial contractility.
• Their release into the plasma is a sensitive marker for cardiac damage, particularly myocardial infarction (MI).

Amylase and Lipase

• Amylase and Lipase, digestive enzymes, are often elevated in acute pancreatitis.

Enzymes as Therapeutic Agents

• Enzymes are used in disease management via various pathways, including dissolving clots (streptokinase & urokinase), treating cancer (asparaginase), improving digestion (pepsin and trypsin), and managing conditions like emphysema (alpha-1-antitrypsin).

Other Useful Enzymes (in Therapy)

• Collagenase breaks down collagen, used in burns.
• Lysozyme, an antibacterial enzyme in tears, is used for eye infections.
• Penicillinase degrades penicillin and helpful for allergic patients.