Jordan D. Rich, B.S.Biomedical Engineering, Univ. of Utah

Presentation Outline:

Primer on enzymes

Enzymatic function in biological systems

Formation of enzyme nanocomplexes

Characterization of form and function

Conclusion: Antidote for alcohol intoxication

Enzymes mediate chemical reactions in biological systems

http://classes.midlandstech.edu/carterp/courses/bio225/chap05/Slide2.GIF

Lowers activation energy and permits unfavorable reactions

Enzymes are polymeric amino acids that assemble into 3-D structures of the lowest free energy

http://www.ebi.ac.uk/training/online/sites/ebi.ac.uk.training.online/files/user/84/images/figure1.png

Structure formation via electrical and hydrophobic interactions

Many possible conformations but only one likely structure (lowest free energy)

REPRODUCIBILITYhttp://www.indiana.edu/~oso/lessons/prot/folding1_files/image002.jpg

Substrates bind to enzyme resulting in conformation change and the breaking/formation of substrate molecular bonds

http://upload.wikimedia.org/wikipedia/commons/thumb/2/24/Induced_fit_diagram.svg/648px-Induced_fit_diagram.svg.png

SubstrateBinding Site

Conformation Change

Bond Reorganization

Increased [H2O2] induces immune activation1 and cell death2

Model of biological enzymatic process: oxidation of glucose

Glucose Oxidase (GOx) oxidizes carboalkoxy group

H2O2 IS A TOXIC INTERMEDIATE

Glucose + 02D-glucono-δ-lactone + H202

Glucose Oxidase (GOx)

OXIDATION

1. Los, M., Droge, W., Stricker, K., Baeuerle, P., and Schulze-Osthoff, K. (2005). Hydrogen peroxide as a potent activator of T-lymphocyte functions. Eur. J. Immun. 25(1):159-165.2. Gardner, A.M., Xu, F., Fady, C., Jacoby, F.J., Duffey, D.C., Tu, Y., and Lichtenstein, A. (1997). Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Rad. Biol. Med. 22(1-2):73-83.

Peroxidases: Nature’s solution to the toxic intermediate problem

Peroxidases employ H2O2 to mediate reduction reactions

Substrate + H202 Product + H20

Horseradish Peroxidase (HRP)

REDUCTION

GOx + HRP = concerted process (reaction + H2O2 disposal)

Mimic concerted process to solve toxic intermediate problem

Mimic cellular membrane to ensure proximity of complimentary enzymes

Perform multi-enzyme-mediated reactions in the extracellular compartment

Design of nanocomplexes accomplished by mimicking nature

Biomimetic: mimic biology

Synthesis of Enzyme Nanocomplexes

1.) HRP & GOx tethered in proximity with inhibitor DNA complex

3.) Inhibitor DNA scaffolds removed

4.) Purification of nanocomplexes: Dialysis with PBS Size-exclusion chromatography

2.) Tethered enzymes encapsulated within polymer network

Monomer:acrylamide

Crosslinker:Bis-methylacrylamide

Transmission Electron Micrograph (TEM) of complexes in solution

Nanocomplex diameter = 30±8nm TEM of nanocomplexes after GOx and HRP labelled with 1.4nm gold particles

Measuring proximity by Förster Resonance Energy Transfer (FRET)

Fluoroisothiocyanate (FITC): @ 450nm excitation, emits at 520nm

Rhodamine B (RhB): @ 450nm excitation, no emission@ 520nm excitation (from FITC emission) emits at 585nm

http://www.molecular-beacons.org/toto/images/FRET.jpg

Fluorosceins:

Requires close proximity (<10nm) for energy transfer

520nm 585nm

(450nm)

<10nm

Spectroscopic determination of enzyme identity within complexes

HRP labelled with RhB

GOx labelled with FITC

Free enzymes(A): no FRET

Indicates that HRP and GOx are within 10nm proximity and that complexes contain both enzymes

Nanocomplexes(B): FRET observed

Polymer encasement not only acts as confining structure but also as protective barrier

Free enzymes and complexes incubated at 65°C (denaturation)

At t=60min, free enzymes lost all function whereas 75% of nanocomplex enzymes maintained functionality

Functional Denatured

HEAT

http://www.ib.bioninja.com.au/_Media/denaturation_med.jpeg

Rate of hydrogen peroxide elimination of Dual-enzyme vs. mono-enzyme analog

Dual-enzyme complex exhibits >900% efficiency of H2O2 elimination over mono-enzyme analog

B

AGOx

+Cat

vsCatGOx +

BADual-enzyme:

Mono-enzyme Analog:

2H2O2

Catalase (Cat)2H2O + O2

Summary of nanocomplex form and function

Average 30nm diameter

Enhanced stability of enzymes

Enhanced function of complimentary enzymes

Excellent prospect as antidote for alcohol intoxication: halt intoxication via enzymatic elimination of ethanol in vivo

Enzyme nanocomplex as antidote for alcohol intoxication

2H2O2

Catalase (Cat)2H2O + O2

CH3CH2OH + 02

Alcohol Oxidase (AOx)

CH3CHO + H202

Employing Alcohol Oxidase:

…and Catalase:

…to eliminate ethanol in vivo

Toxic Intermediate

Loss of intoxicant function

Nanocomplexes exhibit enhanced elimination of ethanol in vivo

Measure of mice BAC at 45min intervals after intoxication with ethanol and administration of placebo (PBS) or enzyme nanocomplex antidote n(AOx-Cat).

Questions ?