WORKING WITH ENZYMES

RS Sangwan
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Introduction
Kinetics of enzyme Reactions
Mechanisms of Enzymes Reactions

Power of Enzyme Reactions

Sturctural Aspects of Enzymes

Practical Aspects of Enzymes Our Selected Work on Plant Secondary Metabolism Enzymes

What are enzymes ?

What is life ? Build-up of new tissues, replacement of old tissues, conversion of food to energy, disposal of waste materials, reproduction, etc But these take place in the face of a paradoxmajority of these biochemical reactions are not spontaneous ! So what makes life possible ? The amazing phenomenon of catalysis. Catalysis is defined as the acceleration of a chemical reaction by some substance which itself does not undergo any permanent chemical change. Catalysts in the biological system are known as enzymes.

Enzymes
an introduction:
The proteinaceous catalysts of biosphere. Typically are very large proteins. Virtually responsible for all the chemical reactions in the cell in which covalent bonds are formed or broken. Make reactions to go at conditions that body can tolerate. Can process millions of molecules every second

Enzyme Properties - In brief.



Enzymes are proteins in nature. High molecular weight molecules made up of chains of amino acids linked by peptide bonds. Molecular weights ranging from 10,000 to 2,000,000. Can be denatured and precipitated with salts, solvents and other reagents. Many enzymes require the presence of other compounds - cofactors (coenzymes, prosthetic groups or metal ion activators).

Some other properties..

Most (bio)chemical reactions have an energy barrier to be overcome (activation energy). On a biochemical level enzymes work by lowering this activation energy of a reaction. Enzymes are classified as Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases and Ligases. Factors affecting enzyme reactions are - temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators.

Plot of substrate concentration versus reaction veloci

Michaelis-Menten equation

A Lineweaver-Burk Plot Lineweaver-

Lineweaver-Burk[1/v] = [K (1)/ V [S] + (1)/V ] m max max equation

Enzyme Inhibition
Inhi it r Binding it T
C etiti e Inhi it r

n nz

Kineti effect
V x is u ch ged; K , s defi ed b [S] required f r 1/2 xi l ctivit , is i creased. K appears unaltered; V ax is decreased proportionatel to inhibitor concentration. Apparent Vmax decreased; Km, as defined b [S] required for 1/2 maximal activit , is decreased.

Nn C etiti e Inhi it r

Un competiti e Inhi itor

Specific ll t the c t l tic site, where it c petes with substr te f r bi di g i d ic equilibriu like pr cess. I hibiti is reversible b substr te. Bi ds r S c plex ther than at the catal tic site. Substrate binding unaltered, but SI c plex cannot for products. Inhibition cannot be reversed b substrate. Binds onl to S complexes at locations other than the catal tic site. Substrate binding modifies enz me structure, making inhibitorbinding site available. Inhibition cannot be reversed b substrate.

Lineweaver- urk Plots of Inhibited Enzymes

Enzyme Isolation & Assay

Pick the enzyme of your interest
(one out of hundreds of thousands)

Purify Assay: 1. End Point Assay 2. Continuous Assay Sensitivity Levels of Detection Colourometric Spectrophotometric Radiometric Fluorescence HPLC/GC Titrimetric

Assay Development: the key to know your enzyme

Rate of decrease in substrate conc. Rate of product formation Coupling with a detectable system Zero Order kinetics Linearity Correct Control Saturating Conditions Temperature and pH conditions Etc. etc.

Designing in vitro enzyme assays...



Choose a starting material / tissue that has a high level of the enzyme of interest such as leaf or flower. flower. Disrupt / break the cells to release the enzyme - such as grinding with liquid nitrogen. nitrogen. Prepare the homogenate in an appropriate Trisextraction buffer - such as 0.1M Tris-HCl (pH 7.0). 7.0). Carry out all extraction and purification steps at 4C - to avoid enzyme denaturation and unwanted proteolysis.

Take your Buffer clues from

   

Available literature, Hit and trial, trial, And last but not the least ..intuition ..intuition ! Remember the goal to have the enzyme in as stable a form as possible.

Other additions to extraction buffer...



Reducing agents - such as DTT or ME. ME. Proteolytic enzyme inhibitors - such as PMSF (a serine protease inhibitor) or EDTA (a metalloprotease inhibitor). Enzyme substrates and cofactors such as magnesium chloride. Polyvinylpyrrolidone (PVP) - to adsorb the phenolics. Sodium azide - as a bacteriostatic agent.

Measuring change in substrate / product concentrations.

Spectrophotometric assays, either in UV or visible assays, ranges - eg. increase in NADH concentration in an assay can be determined by reading change in absorbance at 340 nm wavelength. Fluorimetric assays - if the substrate or product is fluorescent. Luminescence assays - eg. Coupling the firefly luciferase reaction to the reaction of interest. Radiometric assays - if the substrate or product is radioactive. Gas chromatography - if the substrate or product is volatile. HPLC if the substrate or product is soluble in a solvent. solvent.

Carrying out the assay..

 Select an appropriate assay buffer.  Start the reaction by addition of substrate or enzyme. Mix.  Incubate the reaction mixture at a particular temperature - such as 30C - and for a particular time period - say, 15 minutes.  Perform either continuous assay (kinetic) or discontinuous assay (reaction terminated at a fixed time) depending on the method available.  Measure the decrease in substrate concentration or increase in product concentration.  HOW ?

The journey of Enzyme purification..



Do not waste clean thinking on dirty enzymes. enzymes. Kornberg. Remove the insoluble particulate matter - by filtration (through muslin cloth) and centrifugation. centrifugation. Fractionation of protein mixture - by salt (such as ammonium sulphate), organic solvents (such as acetone) or organic polymers (such as PEG). Chromatographic separation of proteins - size exclusion chromatography (such as Sephadex G-100), ion-exchange Gionchromatography (such as Q-Sepharose), hydrophobic Qinteraction chromatography (such as Octyl Sepharose), affinity chromatography, HPLC, TLC, etc. HPLC, TLC,

New Assays Developed at CIMAP Biochemistry Group Tryptophan decarboxylase (TDC) in Catharanthus roseus

Key enzyme for the indole alkaloid biosynthetic pathway Developed a fast fluorescence assay (Analytical
Biochemistry 255:39-46, 1998) 255:39- 1998)

Current assay of choice for biochemical and biotechnological research Allows transient expression analysis Facilitates proteolytic degradation kinetics
(Archives Biochem. Biophys. 2002, Plant Physiol. 2002, BBA 2003, Metabolic Engineering, 2004; J. Biotechnology) Biotechnology)

New Assays Developed at CIMAP Biochemistry Group Terpene Alcohol Acetyl Transferases Key enzyme in volatile ester biosynthesis Developed DTNB based Spectrophotometric assay (Analytical Biochemistry 346: 176-178, 2005)

Secondary Metabolism he
One DMAPP + One IPP One DMAPP + wo IPP One DMAPP + hree IPP .. And so on. GPP FPP

eginning..

Monoterpenes ( 10) Sesquiterpenes ( 15) Diterpenes ( 20)

GGPP

Among these, monoterpenes and sesquiterpenes are volatile and constitute major components of plant essential oils.

Acetate ester formation

Palmarosa Geraniol AAT: Sharma et al.Analytical Biochemistry 346: 176-178, 2005

Palmarosa Geraniol AAT: Sharma et al.Analytical Biochemistry 346: 176-178, 2005

AATs Some properties



Belong to the BAHD family of plant acyltransferases. Two main hallmarks of this family: HXXXD motif (active site) and DFGWG motif (unknown function). Around 60 genes of this family in Arabidopsis. Molecular weights of monomeric enzymes: 40 60 kDa. Usually exhibit wide substrate specificity, both w.r.t. specificity, the alcohol as well as acyl CoA. Also involvement in non-volatile ester biosynthesis: nonphenylpropanoids, alkaloids, other terpenoids, and shikimate pathway derivatives.

THANKS