Enzymes and Enzyme Activity Unit 3

Why do We NEED Enzymes? Enzymes speed up spontaneous reactions Sucrose  Glucose + Fructose ΔG= -7 kcal/mol Spontaneous but SLOW Phosphatase  phosphorylation enzyme – Reaction takes 10 msec How long does it take without the enzyme? -1 trillion years!!!! -Life cannot exist without enzymes

Why are they SLOW!! All reactions require some activation energy (E a ) – Bonds must first be unstable before entering a transition state – Random molecular motion generates enough heat/energy over time to have the reaction Why not use heat? 1)Heat is not site specific; it will speed up everything 2)Too much heat will denature proteins

Catalyst and Reaction Rates Enzymes are biological catalysts – Speed up the chemical reactions – Take part in the reaction but are not changed by it – Regulated by various means to slow, speed up, or stop activity Enzymes, and other catalysts, DO NOT change the ΔG of a reaction! – They DO NOT supply energy to the reaction – They DO NOT make endergonic reactions spontaneous – They DO make the reaction more efficient to the energy already present in not wasted

Lock and Key Formation 4,000 enzymes inside a cell – Why do many? Each enzyme has a specific reactant (substrate) Enzyme specificity: – Most enzymes (locks) have only one substrate (key) – Some have multiple substrates Alcohol dehydrogenase; Alcohol and Antifreeze Active site (keyhole): – Pocket in enzyme that substrate binds to – Specific shape/size for substrate – May have multiple sites

Parts of the Enzyme Induced fit: – Enzyme forces substrate into transition stat – Forms substrate-enzyme complex – 1 or 2 products are then formed and released Enzymes can perform these reactions 100 to 10 million times a second Intracellular enzymes: – Work inside the cell – Work well at 37oC and pH 7 Extracellular enzymes: – Excreted out of the cell to other areas of an organism – Work well in various Temps. and pH

How to Lower the E a Enzymes work in a series of steps: 1)Enzymes, using active sites, force substrates to interact 2)In the active sites, the structure of the enzyme promotes the substrates to enter their transition states (substrate-enzyme complex) 3)As the transition states are reached, the enzyme forces the substrates into their new shape 4)Products are released from the enzyme

Enzyme Reaction Rates Catalase reaction: – Breaks down Hydrogen Peroxide H 2 O 2  H 2 O + O 2 Most enzymatic reactions start at a high rate. Why? – Lots of substrate – Rate only limited by number of enzymes – Initial rate of reaction  slope of tangent of reaction rate Quickly the rate drops. Why? – Less substrate to react

Substrate Concentration Additional reactants (substrates) can speed up product formation – However, at some point, the rate will stop increasing. Why? Saturated – All active sites are full; enzyme is reacting as fast as possible – V max = maximum rate enzyme can work at Why does this not occur when adding more enzymes? – Adding more active sites to make more and more product – Slows when substrate runs low

Enzyme Regulation What are simple methods of enzyme regulation? 1)Temperature – More kinetic energy increases molecular movement – Most enzymes double rate every 10 o C; 0 o C -40 o C – Optimum temp  Animals (37 o C) Plants (40 o C) – Above 50 o C efficiency drops, why? Proteins denature – Taq DNA polymerase: enzyme to make copies of DNA, called PCR, works at 90 o C. Where did it come from? – Thermus aquaticus; a bacterium that lives near thermal jets in the ocean

Enzyme Regulation What are simple methods of enzyme regulation? 2) pH – What pH do most enzymes work best at? pH 7-8, neural – What about secreted enzymes? Depends on the environment – Pepsin, released into the stomach, works best at pH 1.5 – Trypsin, released in the intestine, works best at pH 8

Measuring Reaction Rates If using catalase, how could we measure the rate of reaction? – Measure O 2 produced over time – Measure H 2 O 2 lost over time Measuring rate: 1)Increase in products 2)Decrease in reactants How could we measure the rate of amylase breaking down starch? – Add iodine solution to starch sample – Add amylase – Measure rate of blue-black color disappearance

Enzyme Inhibitors Inhibitor  non-substrate molecule that will lower activity by binding to the enzyme Competitive  – Binds to active site and blocks out substrate – If enough are present, activity stops completely Noncompetitive  – Bind to enzyme at another site – Changes enzyme to lower activity or stop enzyme completely Both forms can be: – Reversible  negative feedback inhibition – Irreversible  cyanide kills humans in seconds

Enzyme Inhibitors Which form of inhibition would effect rates more as substrate concentration decreases? – Competitive inhibition – As less substrate present the chance the inhibitor binds to enzyme increases Why does non-competitive inhibition not vary with substrate competition? – Inhibitor has a separate active site that is not effected by substrates

Enzyme Inhibition In the Body How do most homeostatic levels get regulated in the body? – Negative Feedback Inhibition – Works on enzymes too End-Product Inhibition  – End-Product  final product in a series of reactions – Often acts has an inhibitor for an enzyme earlier in the series – When too much product is made, the end-product shuts down production

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