Effect of Temperature Increasing the temperature increases the energy in the system. Two effects kinetic. denaturing

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1 Effect of Temperature Increasing the temperature increases the energy in the system Two effects kinetic denaturing

2 Kinetic effect Increased motion of molecules Increased collisions between enzyme/substrate Increased rate of reaction Rate 2x increase in rate for every 10 ºC rise in temperature Temperature

3 Denaturing effect Proteins take on the 3-D structure with lowest potential energy - increases their stability Increased energy causes increased motion within the molecule as well as between molecules Weak bonds in the tertiary structure (hydrogen bonds) are broken and new bonds form in different positions New 3-D structures form

4 If the change in 3-D structure alters the active site of the enzyme so that it cannot catalyse the reaction it is said to be denatured % not denatured Temperature

5 Overall effect of temperature Kinetic effect dominant Optimum Denaturing effect dominant Activity Temperature

6 Most enzymes have an Optimum Temperature at which they work best Optimum temperature is not always ~37 ºC Denaturing effect is due to both temperature and time of exposure to that temperature

7 Effect of varying substrate concentration At low substrate concentrations [S] If the number of enzyme molecules [E] remains constant The number of substrate molecules present [S] determines how fast the reaction takes place [Rate of reaction = activity = IRV = v]

8 v V [S] (First order kinetics) [S]

9 At high substrate concentrations [S] If the number of enzyme molecules [E] remains constant The number of enzyme molecules present [E] determines how fast the reaction takes place [Rate of reaction = activity = IRV = v]

10 v V [S] (Zero order kinetics) [S]

11 v V [S]. First order kinetics V [S]. Zero order kinetics Hyperbolic curve - typical of simple enzymes [S] (Michaelis-Menten kinetics)

12 Michaelis-Menten v = V max [S] K m + [S] v = IRV at a specified [S] V max = maximum IRV attainable by the enzyme under given conditions [S] = substrate concentration K m = Michaelis constant

13 v V max K m - [S] at half V max V max /2 Indicator of affinity of enzyme for its substrate High K m - low affinity Low K m - high affinity K m [S]

14 v V max V max /2 Enzyme 1 Enzyme 2 K m K m [S] Both enzymes give same V max Enzyme 1 needs lower [S] to reach V max /2 so has higher affinity for substrate Enzyme 1 has lower K m

15 Plateau on Michaelis-Menten graph only truly reached at infinitely high [S] Cannot carry out experiments at those high concentrations in lab Cannot determine V max and K m experimentally by plotting v against [S] Use a derivation of Michaelis-Menten It is the inverse of the Michaelis-Menten equation Called the Lineweaver-Burk equation

16 Lineweaver-Burk 1/v 1/V max Gradient = K m /V max -1/K m 1/[S] 1 = K m + 1 v V max [S] V max

17 Where is the register?

18 ENZYMES Proteins (biological macromolecules) Catalysts Show specificity Sensitive to changes in physical and chemical environment Their activity can be controlled

19 Enzyme Inhibitors Reduce the rate of an enzyme catalysed reaction Irreversible Reversible Competitive Non-competitive Uncompetitive

20 Irreversible inhibitors Bind irreversibly to enzyme Usually bind via a covalent bond Bind to an amino acid side chain at or near the active site Commonly bind to either Ser (-CH 2 -OH) or Cys (-CH 2 -SH) side chains Binding permanently inactivates the enzyme Usually prevents substrate binding

21 DFP (di-isopropylfluorophosphate) Nerve poison. Ser OH Covalently binds to a Ser residue in acetylcholine esterase H 3 C H 3 C CH O F P O O CH +HF CH 3 CH 3 Prevents breakdown of the neurotransmitter acetylcholine H 3 C H 3 C CH O Ser O P O O CH CH 3 CH 3

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23 F DFP H 3 C CH O P O CH CH 3 CH 3 O CH 3 F Sarin H 3 C CH O P CH 3 CH 3 O Insecticides eg. Malathion, parathion S O H 3 C O P S CH C O C 2 H 5 O CH 2 C O C 2 H 5 CH 3 O

24 Penicillin Antibiotic Covalently binds to a Ser residue in glycopeptide transpeptidase Prevents synthesis of bacterial cell wall peptidoglycan R C O NH C O OH Ser N S CH 3 CH 3 COOH O R C O NH C Ser N H S CH 3 CH 3 COOH

25 Polysaccharide Tetrapeptide Penta-Gly bridge

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27 Competitive Inhibitors Reversible inhibitor Compete with substrate for access to active site Often have structure similar to substrate When bound to enzyme prevents binding of substrate Can be overcome by increasing [S] until it out-competes inhibitor

28 v V max 1/v V max /2 1/V max K m K m [S] 1/[S] -1/K m -1/K m E + S + I EI ES E + P K m increases V max remains unchanged

29 COOH CH 2 COOH Malonate H 2 N COOH CH 2 CH 2 COOH Succinate NH 2 C O COOH CH CH COOH Fumarate para aminobenzoic acid H 2 N NH 2 S O Sulphanilamide

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31 Non-Competitive Inhibitors Reversible inhibitor Bind at a site other than the active site Bind before or after the substrate binds Do not prevent the substrate from binding Prevent catalytic act from taking place Cannot be overcome by increasing [S] Can be removed by repeated dialysis Reaction of the -SH group of Cys with a heavy metal ion eg. Hg 2+, Pb 2+, Ag +

32 v V max 1/v V max /2 V max V max /2 1/V max 1/V max K m [S] -1/K m 1/[S] E + S + I EI+ S ES + I EIS E + P K m remains unchanged V max decreases

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34 Next week Wednesday Surgery 2 Enzymes Thursday 5-6 Assessment practice 1 Proteins and enzymes You will get 25 min to write an answer to the sort of question you might get in the exam in January. There will then be 25 min in which I will go over what the answer should be so you will get feedback on how you performed To help you prepare there are two self-assessment tests available: a paper-based test and an on-line test which you can do as often as you want. Available in the Lecture Support folder on Blackboard