enzyme

ENZYME

Topics Function & Composition ¨  Catalytic Reaction ¨  Enzyme Specificity ¨  Factor affecting enzymatic rate of reaction ¨  Enzyme Regulation ¨ 

Enzyme Function Enzyme: biological catalyst that speed up rates of reaction that would otherwise be too slow to support life ¨  Catalyst: a chemical agent that changes the rate of a reaction without being consumed by the reaction ¨  Enzymes are unaffected by the reaction and are reusable ¨ 

Enzyme Function Enzymes act on substrates at their active site ¨  Substrate: reactant that enzyme acts on ¨  Active site: a pocket where the substrate binds; catalytic center where substrate is converted to product ¨ 

Active Site

¨ 

R-groups on amino acids of the enzyme interact with the substrate at the active site

Enzyme Composition Almost all enzymes are composed of proteins ¨  Exception: ribozyme ¨ 

¤  RNA

that catalyze reactions on other RNA ¤  Hydrolysis at phosphodiester bond

Enzyme Names Most enzymes have an –ase ending ¨  The root name suggests what molecule it acts upon ¨  Example: ATPase ¨ 

Catalytic Reactions Chemical reactions between molecules involve both bond breaking and forming. ¨  Example: sucrose hydrolysis ¨ 

¤  bond

between glucose and fructose is broken ¤  new bonds formed with H+ and OH-

Exergonic Reaction A reaction that releases energy ¨  But reaction still needs an initial investment of energy to break bonds in the reactant ¨  Energy usually supplied in the form of heat (thermal energy) ¨ 

Activation Energy (EA) EA: Amount of energy needed to push the reactants over an energy barrier. ¨  Reactants absorb energy becoming unstable ¨ 

¤  Thermal

agitation increase speed of molecules and number & strength of collisions ¤  Peak of instability = transition state ¨ 

Eventually bond breaks

Change in Free Energy (ΔG) Difference in free energy between products and reactants is the ΔG. ¨  New bonds release more energy than the initial investment to break bonds. ¨ 

¤  ΔG

is negative in an exergonic reaction.

Enzymes lower EA Allows transition state to occur at a lower temperature which speeds up the reaction. ¨  ΔG is unchanged ¨ 

Mechanism for Lowering EA 1.  2.  3.  4. 

Proximity & Orientation Bond strain Microenvironment Covalent Catalysis

1. Proximity & Orientation ¨ 

active site brings reactants closer together and in the correct orientation

2. Bond strain

3. Microenvironment

4. Covalent Catalysis Enzymes may bind covalently to substrates in an intermediate step before returning to normal ¨  Increases reaction rate by: ¨ 

¤  Properly

orienting the substrate ¤  Changing the chemistry at the active site

Enzyme Specificity: Substrate ¨ 

Substrate specific: recognize one specific substrate ¤  can

even distinguish between particular conformation

but the reverse is not true: a given substrate may be acted on by a number of different enzymes ¨  substrate specificity due to the fit between the active site and the substrate ¨ 

Induced-Fit Model As the substrate binds, the enzyme changes shape leading to a tighter fit, bringing chemical groups in position to catalyze the reaction. ¨  Binding of a substrate induces a favourable change in the shape of the active site ¨ 

Animation How enzymes work http://highered.mcgraw-hill.com/sites/0072495855/ student_view0/chapter2/ animation__how_enzymes_work.html ¨ 

Sequence of events E + SàESàES*àEPàE + P ¨  ¨  ¨ 

¨ 

¨  ¨ 

Substrate binds to available active site pocket forming the enzyme-substrate (ES) complex Enzyme changes shape to envelope substrate(s): ES* (transition state) Reaction occurs producing products: Enzyme-product (EP) complex Products lose affinity for the active site: E + P Enzyme is set for another substrate: E + S

Enzyme Specificity: Reaction ¨ 

Reaction specific: Enzyme catalysis is specific for one chemical reaction ¤  Example:

Sucrase is an enzyme that only catalyzes the hydrolysis of sucrose

¨ 

Most metabolic enzymes can catalyze a reaction in both the forward and reverse direction

Group Activity ¨ 

Enzyme Reaction: Sucrase

Factors Affecting Reaction Rate A single enzyme molecule can catalyze thousands or more reactions a second ¨  Factors that affect enzyme activity: ¨ 

¤  Substrate

concentration ¤  Temperature ¤  pH

Rate of Rxn vs. [Substrate]

Why does the increase in rate of reaction slow down at high [substrate]? Example of a saturation curve

Substrate concentration on Rate of Reaction ¨ 

Low substrate concentrations: ¤  Direct

correlation between [S] and rate ¤  á[S],áspeed of binding to active sites, áreaction rate ¨ 

High substrate concentrations: ¤  Enzyme

saturation: active sites on all enzymes are engaged ¤  The only way to increase productivity at this point is to add more enzyme molecules.

**Think: How will the saturation curve change?**

Temperature Effects on enzyme activity átemperature, áspeed of molecules, ácollisions between substrate & active site ¨  Each enzyme has an optimal temperature ¨ 

¤  If

temperature is too high, bonds are disrupted and the protein denatures

pH Effects on enzyme activity Each enzyme has an optimal pH ¨  Most between pH 6-8 ¨  Exception: digestive enzymes ¨ 

¤  those

in the stomach work best at pH 2 ¤  those in the intestine are optimal at pH 8 ¤  both match their working environments.

pH and Enzyme Activity ¨ 

Answer questions on the handout

Enzyme Regulation ¨ 

Inhibition ¤  Competitive ¤  Noncompetitive

¨ 

Allosteric Regulation ¤  Activation ¤  Inhibition

¨ 

Feedback Inhibition

Inhibitors A molecule that binds to a site of an enzyme preventing it from catalyzing reactions. ¨  2 types: ¨ 

¤  Competitive

Inhibitor: binding to active site ¤  Noncompetitive Inhibitor: binding to somewhere other than active site of enzyme

Competitive Inhibition ¨ 

¨ 

Inhibitor binds to the same site as the substrate Think: How do you overcome a competitive inhibitor?

How does a competitive inhibitor change the saturation curve?

Effect of competitive inhibitor on saturation curve

Noncompetitive Inhibition Inhibitor binds somewhere other than the active site ¨  Causes enzyme to become insensitive to substrate concentrations (Conformational change) ¨  The process is reversible ¨ 

Brainstorm ways in which an inhibitor could decrease substrate binding without binding to the active site.

Example of Mechanisms of Noncompetitive Inhibition 1.  2. 

Interference with the active site alters enzyme conformation making the active site unreceptive or less effective

¨ 

Think: Can you overcome noncompetitive inhibition by adding more substrate?

How does a noncompetitive inhibitor change the saturation curve?

Effect of noncompetitive inhibitor on saturation curve

Animation http://www.youtube.com/watch?v=PILzvT3spCQ Competitive and Noncompetitive Inhibitions ¨ 

Allosteric Regulation ¨ 

Allosteric Site: a binding site on an enzyme (not its active site) where regulatory molecules bind ¤  Effector:

regulatory molecule that can activate or inhibit enzyme activity

¨ 

Allosteric Regulation: the binding of effector on the allosteric site causing conformational change of the enzyme. ¤  Allosteric

Inhibition ¤  Allosteric Activation

Effector: Allosteric Regulators Effectors change enzymatic activity by binding weakly to an allosteric site ¨  Allosteric activator: stabilizes the conformation that has a functional active site ¨  Allosteric inhibitor: stabilizes the conformation that lacks an active site (inactive form) ¨ 

¨ 

http://www.springer.com/cda/content/document/ cda_downloaddocument/0602s.swf? SGWID=0-0-45-752220-0

Feedback Inhibition Allosteric inhibitor is a product of the biochemical pathway that it regulates ¨  Feedback Inhibition: Regulation of a pathway by its products ¨ 

¤  Products

bind to the enzyme that catalyze the first reaction of the pathway

Animation Feedback Inhibition of Biochemical Pathways http://highered.mcgraw-hill.com/olcweb/cgi/ pluginpop.cgi?it=swf::535::535::/sites/dl/free/ 0072437316/120070/bio10.swf::Feedback %20Inhibition%20of%20Biochemical%20Pathways ¨ 

HW Questions 1. 

2. 

Compare noncompetitive inhibition and allosteric inhibition. Compare allosteric inhibition and feedback inhibition