2-12 Enzyme Regulation and Clinical Applications Flashcards
What is allosteric regulation?
Binding at an allosteric site changes conformation of the enzyme so that binding to other sites is affected. Allosteric enzymes also:
- Freq. operate at control pts in met. pathways
- Often have multiple active sites, subunits
- Are modulated either by the levels of their own substrate or by the levels of other activating/inhibitory modulators
- Do not follow Michaelis-Menten kinetics
- Are categorized as heterotropic (not substrates, do not bind at active site) or homotropic (substrate itself is an effector)
What kinetics do allosteric enzymes follow?
NOT Michaelis-Menten kinetics.
- Allosteric enzymes show an S-shaped curve rather than a rectangular hyperbola
- K0.5 = concentration of substrate giving half-maximal activity
- Allosteric modulators can affect either K0.5 or Vmax and can be either activators or inhibitors
What is regulation by reversible covalent modification?
Reversible covalent modification can alter enzyme activity by:
- Causing a conformational change that affects catalysis
- Altering cellular localization of the enzyme
- Altering interactions with other proteins
Common reversible covalent modifications:
- phosphorylation
- methylation
- acetylation
- glutathionylation
- addition of ubiquitin and ubiquitin-like molecules
What is regulation by irreversible covalent modification?
Enzymes are synthesized in an inactive form and activated irreversibly in the time and/or place where they are needed.
ex) Zymogen = inactive precursor form of a protease that is activated by a specific proteolytic cleavage by another protease (e.g., pepsin, chymotrypsin, thrombin, caspases, etc.)
How is enzyme regulation involved in the activation and regulation of the digestive enzyme protease cascade?
Digestive enzymes are synthesized as zymogens and stored in pancreas, then activated upon release into the small intestine.
How is enzyme regulation involved in the activation and regulation of the coagulase cascade?
Maintenance of blood volume requires three rapid responses to blood vessel injury:
- Rapid activation of blood coagulation: Zymogen activation cascade allows sequential activation of a series of serine proteases. Each activated protease can activate many target proteases, so signal is amplified (irrev. covalent mod.). Can be inhibited by serpins like anti-thrombin, which binds with the help of heparin.
- Localization of clot to site of injury: Glutamate residues are modified to g-carboxyglutamate on several of the serine proteases of the cascade in a vitamin K-dependent pathway. Ca2+ bridges the modified proteins and the membrane at the site of injury (rev. covalent mod.)
- Rapid termination after clot formation to prevent thrombosis: Reverse zymogen activation cascade hydrolyzes clot
(irrev. covalent mod.)
What is regulation by protein-protein interactions?
Reversible or irreversible inhibition of enzyme activity by other proteins. Signal transduction cascades are often regulated by protein-protein interactions. Examples include:
- Serpins like anti-thrombin
- Protein kinase A: regulated by interaction of cAMP with regulatory subunits
- Calmodulin: shows Ca2+-dependent interactions with multiple enzymes
Why are enzymes used as diagnostic tools?
- Diagnostic measurement of enzyme levels
- Measurement of substrate or metabolite levels
- Diagnosis of tissue damage or tumors by isozyme distribution (tissue enzymes are present in plasma because of tissue damage or overproduction)
What are the general principles of clinical enzyme assays?
Use an enzyme specific for the substance being measured.
In order to determine how much enzyme is present, clinical labs determine the rate of product formation with time, either of the direct reaction product or through a coupled enzyme system in which one of the products of the first reaction is stoichiometrically converted to a detectable product by a second reaction.
Conduct the assay at saturating [substrate] (i.e., at Vmax), because the rate of reaction will be directly proportional to the amount of enzyme present.
Be aware of confounding factors in the sample that may interfere with enzymatic activity.
What is the difference between serum and plasma?
Plasma is the fluid part of blood that surrounds various cell types (physiological fluid).
Serum is the liquid that remains after whole blood is coagulated and centrifuged (primarily used for laboratory assays).
What are the principal serum enzymes used in clinical diagnosis?
Think CLAPLAP.
- Creatine kinase: muscle disorders, MI
- Lactate dehydrogenase isozyme 5: liver diseases
- Alanine aminotransferase (ALT/SGPT): MI, also liver
- Phosphatase, acid: metastatic prostate carcinoma
- Lipase: acute pancreatitis
- Amylase: acute pancreatitis
- Phosphatase, alkaline (isozymes): various bone disorders, obstructive liver diseases
What are isozymes?
Different forms of an enzyme that carry out the same reaction. They . . .
- Have diff. AA sequences, diff. chemical properties, and diff. enzymatic characteristics
- May have a specific expression in diff. tissues, or a specific pattern of expression during development
Diff. isozymes can be distinguished by:
- charge differences (electrophoresis)
- specific monoclonal antibodies
- differences in enzymatic properties or inhibitor sensitivities
