POST LAB ENZYME CATALYSIS LAB AP Lab #2 AP Biology

Let’s review the main concepts and vocabulary! FIRST…. Let’s review the main concepts and vocabulary!

What is an enzyme? A specific type of protein Speeds up (catalyzes) chemical reactions LOWERS activation ENERGY Are NOT used up during rxns instead are recycled/reused Have primary, secondary, teritary, and (sometimes) quartnary structures Can be denatured

http://www.stolaf.edu/people/giannini/flashanima t/enzymes/transition%20state.swf

What are the 4 conformational structure of a protein? Primary LINEAR amino acid sequence Peptide bonds hold protein together Secondary Alpha helices and Beta pleated sheets Hydrogen bonds hold together this level Tertiary More bonding/protein folding  creates 3D shape Ionic bonds, H bonds, Van der Waals forces, disulfidge Quartnary 2+ polypeptides put together (makes bigger proteins and enzymes)

http://www.stolaf.edu/people/giannini/flashanimat/proteins/protein%20structure.swf http://www.hippocampus.org/Biology;jsessionid=1639F9E4709EA30A7D60337870E663BF BIOMOLECULES ANIMATION: Structure/Function of Proteins

WHAT DO THE “R GROUPS” DO IN PROTEINS? Each amino acid has UNIQUE R-groups attached to their carbon skeleton Involved in secondary/tertiary structure bonding Some R groups create: Nonpolar Amino Acids (NO CHARGE!!!) Polar Amino Acids (+ or – CHARGE)

Enzyme Vocabulary Substrate = ? Active Site = ? Induced fit = ? SPECIFIC reactant that a SPECIFIC enzyme binds to Active Site = ? Area on enzyme which binds to substrate Induced fit = ? Occurs in enzyme-substrate complex  enzyme binds substrate “tighter” leading to quicker transition state Weakens substrate bonds  lowers EA

ACTIVE SITE IN AN ENZYME http://highered.mcgraw- hill.com/sites/0072943696/student_view0/chapter2/animation__how_e nzymes_work.html

WHAT IS “DENATURATION” AGAIN? When a protein unravels and loses its native conformation/shape 3D shape (teritary) and secondary structures are disrupted! Enzyme function is lowered or stops http://highered.mcgraw-hill.com/sites/0072943696/student_view0/chapter2/animation__protein_denaturation.html

WHY DOES PH DENATURE PROTEINS? BASE excess OH- ions ACID = excess H+ Protein's shape is altered Active site is blocked Enzyme cannot bind substrate and make productes

WHY DOES TEMPERATURE DENATURE PROTEINS? Kinetic energy changes Atoms move differently  affects bonds in protein A higher temperature generally results in increase activity b/c molecular motion increases resulting in more molecular collisions If, however, temp rises above a certain point, the heat will denature  molecules move too fast and can’t H-bond Cold temp’s SLOW DOWN or stop activity b/c molecular motion decrease

[SUBSTRATE] ALSO EFFECTS ENZYME ACTIVITY If [ ] of enzyme is constant… at lower [substrate]  [substrate]= limiting factor As [substrate] increases, RATE of enzyme activity also increases However, at very high [substrate]  enzymes become saturated with substrate and a higher concentration of substrate does NOTHING to increase the reaction rate All the enzymes are already in use

GRAPHS

Lab #2: Enzyme Catalysis POST LAB… Lab #2: Enzyme Catalysis

+ O=O What is the substrate used in lab= ? H2O2 (hydrogen peroxide) What is the enzyme used in lab= ? Catalase What are the products used in lab= ? H2O (water) and O2 (oxygen gas) CATALASE + O=O Oxygen gas

NOTE: Oxygen gas(O2) forms BUBBLES ENZYME SUBSTRATE (REACTANT) PRODUCTS

WHY DOES H2O2 NEED TO BE BROKEN DOWN? H2O2 is poisonous (TOXIC) to cells! H2O2 is naturally made during cellular respiration (ATP production in cells)

REVIEW… PART A What did the catalase solution do when H2O2 was added? Why? O2 bubbles formed Catalase broke down H2O2 What did the liver and potato tissue do when H2O2 was added? Why? Catalase broke down H2O2 in BOTH tissues What did the boiled liver do when H2O2 was added? Why? High temp’s denatured catalase enzyme function stopped NO bubbles (O2)

REVIEW… PART B What did we establish a BASELINE? We needed to know HOW much H2O2 is in 3% solution THIS IS OUR INITIAL AMOUNT OF H2O2 We put this amount of H2O2 is each cup to begin with What did we use KMnO4 (potassium permanganate)? Amount of KMnO4 = Amount of H2O2 LEFT inside cup

We used INITIAL and FINAL readings to determine TOTAL amount of KMnO4 in cup (equal to amount of H2O2)

TITRATION RXN The Titration Reaction is below: 5 H2O2 + 2 KMNO4 + 3 H2SO4  K2SO4 + 2 MnSO4 + 8 H2O + 5 O2 ** H2O2 reacts with 2 KMNO4 ; once H2O2 all used up KMNO4 can’t react anymore

REVIEW… PART D Why did we use HCl (hydrochloric acid)? The hydrochloric acid (HCl) will “freeze” enzyme reaction. WHY??? Adding H+ ions  disrupts/BLOCKS active site Which cup should have the MOST H2O2 LEFT (least amount of H2O2 decomposition)? 0 sec cup. Why? Enzyme had no time to work Which cup should have the LEAST H2O2 LEFT (most amount of H2O2 decomposition)? 360 sec cup. Why? Enzyme had most time to work

RESULTS: ANALYSIS OF DATA You need to: Calculate the RATE OF ENZYMATIC REACTION

GRAPHS

CONCLUSIONS Enzyme reaction rate is affected by: pH, temp, [substrate], & [enzyme]

SOME REAL-LIFE APPLICATIONS…. Bacterial enzymes and use of disinfectants Many disinfectants, such as chlorine, iodine, iodophores, mercurials, silver nitrate, formaldehyde, and ethylene oxide, INACTIVATE bacterial enzymes and block metabolism. Extremes of temperature to control bacteria. High temperatures, such as autoclaving, boiling, and pasteurization, denature proteins and STOP functions Cold temperatures, such as refrigeration or freezing, SLOW DOWN or STOP enzyme rxns

ENZYME TUTORIAL ANIMATION http://www.hippocampus.org/AP%20Biology%20II Principles of Bioenergetics View Part 1, 2, and 4