Killing bacteria by inhibiting their enzymes. BIOCHEMISTRY LAB

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1 BIOCHEMISTRY LAB CHE554 Enzyme Kinetics Motivation: Enzymes mediate almost all the chemistry in cells, and thus in life. They are the best catalysts known and have applications in every field as tools for synthesis, remediation, chemical recycling and intervention targets. Killing bacteria by inhibiting their enzymes. While human actions or accidents produce injuries, death is very often dealt by bacteria. Up to 90% of the native peoples of America died of European and African diseases even before colonists vied for the land. Approach: a chromogenic reaction catalyzed by β-galactosidase is used to visualize the effects of substrate concentration and inhibitors of different types on enzyme-catalyzed reaction velocity. Experiment #7, pages , Population_history_of_indigenous_peoples_of_the_Americas

2 Transpeptidase s essential role Bacteria resist cell lysis by virtue of their cell walls. The cell wall is constructed of peptidoglycan produced by enzymes. Transpeptidase forms and remodels the peptide crosslinks between layers of peptidoglycan. Penicillium notatum s defense 3! P. notatum exports a secondary metabolite we call penicillin, which disrupts cell wall synthesis and leads to bacterial death. Penicillin was our first wonder drug and has saved many lives.! However evolution is a live and well, bacteria have evolved. 4

3 Penicillin inhibits one enzyme Augmentin: An enzyme inhibitor and an inhibitor of the enzyme that degrades it. Amoxicillin inhibits transpeptidase. but is degraded by another β lactamases can hydrolyze the reactive lactam ring of penicillin and related antibiotics. 5 Clavulanic acid inactivates many β lactamases. It is the additional ingredient in augmentin. As enzymes gain use in consumer products, remediation strategies, synthesis and more, we need to be able to characterize their activity and factors that interfere with it. 6

4 New antibiotics for new bacteria Background (Enzyme Kinetics)! Enzymes influence the rate at which equilibrium is obtained, but not the equilibrium position of the reaction.! Kinetics, in this context, is the study of reaction product formation as a function of time. The rate of product formation is a measure of the reaction velocity. S P v = d([p]) dt = d([s]) dt Targeting virulence: a new paradigm for antimicrobial therapy Anne E Clatworthy, Emily Pierson & Deborah T Hung Nature Chemical Biology 3, (2007) Published online: 20 August 2007 doi: /nchembio _F1.html v = reaction velocity

5 Biology needs reactions that are not naturally fast, because it needs stable compounds. Enzymes accelerate reactions by up to x Wolfenden and Snider (2001) Acc. Chem. Res. 34: Wolfenden & Snider (2001) Acc. Chem. Res. 34(12):

6 Reaction equilibrium vs. velocity. Concentration [S]0 Rate-limiting step and transition state energy.! The reaction is accelerated by lowering the free energy of transition state activation. [P]eq Keq = [S]eq kvel = kbt h e-δg /RT [S]eq [P]eq Keq = e -ΔG /RT [P]0 d[p]/dt = v v0 = k 3[ET] [S]0 KM + [S]0 time ΔF = ΔG = -RT ln Keq! We measure the initial reaction velocity, when [P] 0 and [S] [S]0. 11

7 Observed Initial Rate of Reaction Background (Michaelis-Menton)! The Michaelis-Menton equation describes the relationship between initial velocity (v o ) and initial substrate concentration ([S]0 ). measure and plot initial velocity (v o ) as a function of substrate concentration ([S]0). Initial velocity is used to eliminate effects of product buildup (slowing down reaction). At some point adding more substrate does not further increase the reaction rate. A plateau is observed corresponding to saturation of the enzyme. For single substrate, V max occurs when all of substrate is in ES state. Figure II-8 K M (the Michaelis constant) is equal to the substrate concentration that yields v o equal to V max /2. This constant is used as a general measure of the stability of the enzyme substrate interaction. K M has the form of dissociation constant. The analogy is best when k3 << k2. Officially; K M = (k 2 + k 3 ) / (k 1 ) (More generally K M = (k- 1 + k 2 ) / (k 1 ) ) Vmax is the velocity achieved when all enzyme is saturated with substrate.

8 Lineweaver-Burke! The double-reciprocal plot (Lineweaver-Burke plot). Easily calculate K M and V max by simply rearranging Michaelis-Menton equation plot 1/[S] versus 1/v o Still need to measure initial velocity (v o ) as a function of substrate concentration (S). Inhibition! To combat disease we inhibit enzymes.! Which kind of inhibition will be most effective (what type of inhibitor would you like to design)? Competitive. Non-competitive. Uncompetitive. 16

9 I E EI Competitive inhibition K I S ES E + P Competitive inhibitors.! Competitive inhibitor binds to the free enzyme in place of the substrate. More substrate is needed to get the same rate, and maximum rate doesn t change.! One can see and measure inhibition by plotting 1/[S] versus 1/v o as a function of inhibitor concentration. v o = V max [S]/(K M + [S]) becomes v o = V max [S]/(K M + [S]), K M = K M (1+[I]/K I ) Vmax is not changed. S and I compete for E. Any ES formed reacts at the same rate as in the absence of inhibitor. From CHE550

10 Noncompetitive inhibition S E I EI K Ie ES E + P I K Is ESI v o = V max [S]/(K M + [S]) becomes v o = V max [S]/(K M + [S]), V max = Vmax/(1+[I]/Kie) K M = K M (1+[I]/K Is )/(1+[I]/Kie) PURE noncompetitive: K Ie = K Is I E EI K Ie S I ES ESI K Is E + P K Ie = K Is Inhibitor binding does not affect the E ES equilibrium v o = V max [S]/(K M + [S]) becomes v o = V max [S]/(K M + [S]), K M = K M V max = V max / (1+[I]/K Is ) From CHE550 From CHE550

11 Noncompetitive Inhibitors.! Noncompetitive inhibitor binds to free enzyme or enzyme-substrate complex. Notice how the curves differ from that for a competitive inhibitor. Uncompetitive inhibition E S I ES K I IES E + P Pure non-competitive, Mixed non-competitive Figure II-11 I binding affects both the equilibrium among E states and V max. v o = V max [S]/(K M + [S]) becomes v o = V max [S]/(K M + [S]), K M = K M /(1+[I]/K I ) V max = V max / (1+[I]/K I ) From CHE550

12 Uncompetitive Inhibitors! Uncompetitive inhibitor binds only to the enzymesubstrate complex. β-galactosidase Lactose glucose + galactose. Homotetramer. Product of the lacz gene. The protein can be expressed in two parts. laczα and laczω. Catalytic activity requires both. 24

13 The Experiment (1) Two Experiments! We will employ the enzyme β-galactosidase, which allows lactose metabolism in the bacterium Escherichia coli.! We will use o-nitrophenyl-β,d-galactopyranoside (ONPG) instead of lactose because it is hydrolyzed into a color-containing solution (so we can monitor the reaction).! We will study its kinetics in the absence and presence of inhibitors. This will be done using time-dependent spectroscopy.! Day 1: Determine the enzyme concentrations that produce linear kinetics. This concentration will be used for Day 2 experiments Steps 1-7, Determine Enzyme Activity Make the several dilutions of enzyme before beginning to use the spectrophotometer. Steps 8-10, data analysis Calculate the extinction coefficient of ONP. Day 2 Kinetic parameters K M, Vmax and KI: Steps 1-5, Determine K M and V max Steps 6-9, data analysis Steps 1-4, Inhibitor Effects on Activity Steps 8-10, data analysis (do one inhibitor only) Chromogenic substrates! The following page lists the people who should use MGP and those who should use MTG.

14 Section 1 Section 2 A.-F. Miller 2013 Inhibitor to use Name Locker Group Inhibitor Danielle Edwards L41C B MTG An Lien Ho L35C A MGP Joanna Ng L35C B MGP Niranjana Warrier L44C B MTG Kaeto Vin-Nnajiofor L34C A MTG Morgan Sizemore L34C B MGP Corey Lee L31C A MGP Matthew Wolfe L31C B MTG Derrick Lewis L28C A MTG Travis Johnson L38C A MGP Jessime Kirk L38C B MGP Carl Archemetre L44C A MTG Falak Patel L35C B MTG Robert Reed L41C B MGP Dylan Woolum L35C A MGP Kalen Wright L41C A MTG 27 Changes From the Book! We will follow the experimental protocol exactly for Days 1 and 2 except Skip steps on Day 1 (determination of enzyme concentration) use either MGP, or MTG on Day 2 (see prev. pg. for who should do which). omit steps on Day 2.! We will not be doing Days 3 and 4.! Technical tweaks Do not vortex enzyme-containing solutions. Invert or swish in and out of a pipettor. Start a timer as you add enzyme. Record the time at which you make your first absorbance reading if it is not exactly at 30 sec. Do the same for any time point that is not at the target time. Work with a buddy who can adjust your pipettor for the next addition while you work with the spectrophotometer. For step 10, first plot activity vs. enzyme amount present, and use only points that fall in a linear regime, when calculating your stock solution activity.

15 Hint for managing the analysis. For step 10 you will see: curve. Use the following equation to determine the galactosidase activity (micromoles of ONPG hydrolyzed per minute/per milliliter of enzyme) in each reaction: Activity ( A 420/ time)(1.25/ A 420max ) V p The purpose of this equation is to convert ΔA420 to concentration of product formed. i.e. we need an extinction coefficient! We will use the imperfect solution of assuming that the timecourse you allowed to go to completion converted ALL substrate to product. Therefore the final (max) [P] = initial [S] = 0.25 mm (show how I got this). Data Analysis Run your long time course with a buddy, each of you can put a tube in the same spectrophotometer and you can each take turns reading absorbances 29 ε = ΔA420, max / 0.25 mm. C(t) = ΔA420(t)/(ΔA420 / 0.25 mm) You made 5 ml of solution. Thus the number of moles of P you formed is 5x10-3 L x ΔA420(t)* 0.25 x 10-3 mole L -1 /ΔA420,max P = 1.25 x10-6 moles x ΔA420(t) /ΔA420,max

16 Data Analysis Example Data from Day 1 A420 Prepare a table in advance so that during the experiment you will be filling in boxes with absorbances (also allow a column for time of observation). Write your results down in real time. Enzyme Activity at One Concentration A420 Look for linearity over 2-4 minutes

17 Linear regime for enzyme activity! For point 10, instead of averaging, use the slope of the linear range to calculate activity on a per-volume basis (volume of stock solution). Experimental Considerations! Pay careful attention to which steps have experiments and which have data analysis. You can do the data analysis later. related to slope of A420 vs. time! Make sure your data is saved, for example to the hard drive of one of the computers, in case your memory stick goes bad.! Delete all old files from the spec. to free up more experiments for use.! It will be easy to confuse substances, so please be careful. related to 1/dilution 33

18 Safety Considerations! Observe all normal laboratory safety practices.