Enzyme kinetics. Irreversible inhibition inhibitor is bound tightly to enzyme - very slow dissociation can be covalent or non covalently bound

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1 Enzymes can be regulated by acceleration and inhibition inhibition very common - several different mechanisms competitive / non competitive reversible / irreversible

2 Irreversible inhibition inhibitor is bound tightly to enzyme - very slow dissociation can be covalent or non covalently bound

3 Competitive inhibition is always reversible inhibitor resembles substrate - competes for binding sites at enzyme reduces rate of catalysis by reducing number of molecules bound to enzyme can be overcome by increasing substrate concentration

4 Noncompetitive inhibition is always reversible inhibitor binds to enzyme at same time as substrate - at different binding site does not reduce the number of substrate molecules bound to enzyme molecules but the turnover rate of each enzyme molecule not affected by increased substrate concentration

5 Competitive inhibition:

6 Non competitive inhibition:

7 Dissociation constant for competitive inhibitor is: The Km also changes in the presence of an inhibitor. New Km is call apparent Km Is equal the real Km modified by inhibitor concentration and inhibitor dissociation constant

8 Non competitive inhibition does not change the Km. Only Vmax is changed Concentration at which Vmax is reached is not changed same Km

9 By adding possible (irreversible) inhibitors one can investigate possible active sites

10 Remember transition states? transition state analogues are powerful inhibitors transition states bin usually very sellectively and very tightly to active sites e.g. proline racemase (changes conformation of proline) is in inhibited by Pyrole 2-carboxylic acid competitive or non competitive?

11 Very useful tool is a double reciprocal plot Lineweaver-Burk plot Easy way of getting Km and Vmax from a plot substrate concentrations and speed

12 Lineweaver-Burk plots make distinction between competitive and non competitive inhibition easy different slopes but same y intercepts mean competitive inhibition different slopes and same x intercept mean noncompetitive inhibition

13 Lineweaver-Burk for competitive inhibition includes the inhibitor dissociation constant: Ki is also the difference in slope between the inhibited and uninhibited speed over concentration plot

14 In non competitive inhibition the factor by which the apparent Vmax is increased is equal to the factor increasing the slope

15 Problems: Determine Vmax and Km for both cases What type of inhibition is this? Inhibitor with a concentration of 2mM was used. what is its binding constance?

16 Problems An kinetics experiment with several concentrations of the same enzyme was performed. which graphs would you expect as the result and why?

17 Problems An inhibition experiment was performed draw the lineweaver-burk plot for the following data What could be the reason for decreased velocity at higher substrate concentrations?

18 Problems assume an enzyme is inhibited in this way what Michaelis Menten curve would you expect and why draw the corresponding Lineweaver-Burk plot

19 Remember these assumption for MM: concentration of the enzyme is very small compared to the concentration of the substrate substrate is not limiting (there is enough substrate - but concentrations may vary) amount of product is small compared to the amount of substrate available. Enzyme concentration does not change

20 Advice: kcat/km (in 1/sM) often called catalytic efficiency write down all MM related equations use a flash card A cheat sheet will be provided in the quiz

21 Don't want to draw but calculate? y=mx+b first calculate m: take any point and use formula : set x=0 for the y intercept set y=0 for the x intercept