Lect 13_Enzymes ID 7160 SK PDF

Foundation Biochemistry Enzymes ID# 7160 ID# 7162 Dr. Selma Alkafeef Biochemistry Department  Enzymes are very powerful and efficient catalysts Enzymes: – Proteins that act as biological catalysts and facilitate specific reactions – Accelerate...

Foundation Biochemistry Enzymes ID# 7160 ID# 7162 Dr. Selma Alkafeef Biochemistry Department  Enzymes are very powerful and efficient catalysts Enzymes: – Proteins that act as biological catalysts and facilitate specific reactions – Accelerate reaction rates (by up to about 1014 to 1016) – Without enzymes, reactions would not occur at a sufficient rate to sustain life Enzymes are very powerful and efficient catalysts 78 million years vs 18 milliseconds! A. Radzicka and R. Wofenden. Science 267 (1995):90–93.  Enzymes are very powerful and efficient catalysts Enzymes: – Proteins that act as biological catalysts and facilitate specific reactions – Accelerate reaction rates (by up to about 1014 to 1016) – Without enzymes, reactions would not occur at a sufficient rate to sustain life – Reduce or prevent generation of unwanted products from particular reactants – Work in an organized sequence to make particular products (metabolic pathway) – The enzyme is not consumed during the course of the reaction – Can NOT change the free energy (∆G) of a reaction  General properties of enzymes Molecular weight of enzyme polypeptides Glucose is highly variable – Ranges from about 12,000 to over 1 million Da; enzymes are relatively large molecules Catalytic activity requires native protein conformation – denaturation will decrease or destroy catalytic activity Localization is important: Enzymes in the same pathways compartmentalized together (i.e. the mitochondria) Compartmentalized to prevent unwanted activity (lysosome)  Cofactors: enzyme add-ons Enzymes may require additional chemical components for catalytic activity. Cofactors can be: – Inorganic ions such as Zn2+, Mg2+, Mn2+, Fe2+ – Coenzymes: complex organic molecules such as NAD+ – Organometallic, i.e. heme – Permanently bound: prosthetic groups (i.e. heme on hemoglobin) apoenzyme + cofactor = holoenzyme Cofactors can assist in a reaction – Directly by forming covalent interactions with substrate – Oxidation-reduction: have unique functional groups that accept or donate electrons – Non-catalytic roles: influence enzyme tertiary structure Have ALMOST NO catalytic activity and specificity without enzymes! Defining enzyme amounts by activity For many samples, especially clinical samples (for example in a sample of blood plasma), the actual molar amount (number of molecules) of a particular enzyme is unknown. The amount of enzyme is reported in terms of activity. Enzyme activity, standard unit Specific activity  Enzyme activity: standard unit A standard unit has been established by the International Union of Biochemistry: One International Unit (IU) of an enzyme is the amount that catalyzes the formation of one micromole of product in one minute 1 U = μmol/min at the given conditions! Enzymes are sensitive to factors such as pH, temperature and ionic strength, so the particular conditions of the assay must be specified  Enzyme activity: standard unit One International Unit (IU) of an enzyme is the amount that catalyzes the formation of one micromole of product in one minute 1 U = μmol/min Inherent characteristic of the enzyme at the given condition In order to generate 1 μmol in 1 minute: you need more total amount of a low activity enzyme but less total amount of a high activity enzymes  Enzyme activity: specific activity Specific activity: Units of enzyme activity per milligram of protein in a sample SPECIFIC ACTIVITY = UNITS OF ENZYME ACTIVITY / MASS OF PROTEIN 1 U = μmol/min U/mg μmol/min/mg Standard unit Standard unit/mg Specific activity is a measure of enzyme purity  Enzyme activity: specific activity Example: The specific activity of the isolated enzyme was measured at 150 µmoles/min/mg protein before purification and 800 µmoles/min/mg after purification Purification Only purple enzyme Total mg Total mg protein higher protein lower Less pure, less specific activity More pure, higher specific activity  Isoenzymes Isozymes are different forms Isozymes may differ in: of an enzyme that catalyze – sub-cellular location the same chemical reaction – tissue distribution – kinetic properties (e.g. affinity Gene A Gene B Gene C for substrates and inhibitors) – regulatory properties (different isozymes may respond to different cell signals e.g. Isozyme A Isozyme B Isozyme C through effects of phosphorylation or other modification affecting activity) – cofactor used (e.g. NAD+ or NADP+) CK1 – BB (brain) Clinical application of CK2 – MB: (cardiac) CK3 – MM: (muscle) isoenzymes Within heart: Appearance of tissue-specific CK2 – MB: 33% isozymes in blood plasma can be a CK3 – MM: 66% useful diagnostic marker for tissue damage in particular diseases Example (Lippincott text fig. 5.22): Myocardial muscle contains creatine kinase as the CK2(MB) isozyme. This isozyme is not released in large quantities by damage to other tissues. Skeletal muscle and brain have a different isoforms Non-protein biological catalysts Enzymes are biological catalysts that are proteins Ribozymes are another type of biological catalyst made up of RNA - ribosomes Enzymes can also be made of both proteins and other components like RNA (ribonucleoproteins) - telomerase How do enzymes catalyze reactions?  How do enzymes work as catalysts? Enzyme (E) with Substrate (S) : E + S ⇌ ES ⟶ EP ⟶ E + P An enzyme provides a special environment for a particular chemical reaction Reactions occur within a specific region (pocket) termed the active site Reactants (substrates) bind to the active site and products are released from the site when the reaction has occurred Properties of the active site determine the selectivity of the enzyme for substrates and the efficiency of catalysis  How do enzymes accelerate chemical reactions? * Transition state Lowering the * energy barrier accelerates the reaction Enzymes do not change the free A+B energy of a reaction (∆G) or the P+Q equilibrium of a reaction DOES change the rate at which equilibrium is Uncatalyzed A+B P+Q reached Catalyzed  Properties of the active site Size and shape: binds substrate(s) but excludes unwanted molecules Polarity/hydrophobicity/electrical charges promotes high affinity binding of substrate Chemical groups necessary for catalytic activity in correct locations and orientations (and binding sites for cofactors, if required)  Models for substrate-enzyme interactions Lock and key Induced fit STATIC model DYNAMIC model  Catalysis in the active site Reaction proceeds: Reactant(s) ⇌ Transition state ⇌ Product(s) The transition state has a structure different from both substrate and product Substrate bonds are maximally strained in the transition state → high energy state Active site binds the transition state more tightly than either reactant(s) or product(s) How do enzymes work as catalysts? How do enzymes work as catalysts? Enzymes are very efficient catalysts because they: 1. Bring substrate(s) and catalytic groups together (proximity effect). 2. Hold substrate(s) at the exact distance and in the exact orientation necessary for rapid reaction (orientation effect). 3. Provide acidic, basic, or other types of groups required for catalysis (catalytic effect). 4. Lower the energy barrier by inducing strain in bonds in the substrate molecule and stabilizing transition state (energy effect).  Serine protease mechanism of catalysis Orientation & Catalytic & energy Catalytic & energy proximity effects effects effects 1 2 ∆G ↑ 3 ∆G ↓ Phe Ser, His, Asp: Tetrahedral oxyanion intermediate: H transferred to C terminal catalytic triad Transition state complex peptide fragment  Serine protease mechanism of catalysis Orientation & Catalytic & energy Catalytic & energy proximity effects effects effects 4 5 ∆G ↑ 6 ∆G ↓↓ Acyl bond cleaved; Second tetrahedral oxyanion H2O enters active site H back to serine intermediate N terminal fragment released What conditions can affect enzyme catalytic activity?  Effects of pH on reaction rate pH optimum There will be an optimum pH or range of pH at which enzyme activity is greatest Recall: amino acids have different ionization states depending on isoelectric point (pI) and pH pH favors native protein structure and optimum substrate binding pH affects catalytic activity (active site ionizable groups in favored Rate of reaction against pH for the enzyme fumarase ionization state)  Effects of pH on reaction rate pH optimum There will be an optimum pH or range of pH at which enzyme activity is greatest Recall: amino acids have different ionization states depending on isoelectric point (pI) and pH pH favors native protein structure and optimum substrate binding pH affects catalytic activity (active site ionizable groups in favored Rate of reaction against pH for the enzyme fumarase ionization state) Effects of temperature on reaction rate Increasing temperature increases reaction rates Enzyme (increased kinetic energy of denaturation substrates) High temperatures distort enzyme structures (disturb secondary and tertiary structure) and decrease catalytic activity Higher temperatures may denature the enzyme Theoretical temperature profile for a simple enzyme Effects of substrate concentration on reaction rate Reaction rate low Reaction rate high Thank you!