BCOR 011 Lecture 12 9282005 ENZYMES Last time

BCOR 011 Lecture 12 9/28/2005 ENZYMES

Last time… - G reaction “can” go spontaneous But when will it go? And at what rate?

Thermodynamics Whether a reaction will occur Kinetics WHEN a reaction will occur

What governs WHEN a reaction will occur? The tower of blocks falling is favorable but when will it happen? Oxidation of carbohydrate polymers (starch) to carbon dioxide and water is favorable but when will it happen? Gasoline burning to carbon dioxide and water is favorable but when will it happen ?

For a Reaction to occur need to Destabilize Existing State to INPUT ENERGY Destabilization energy input “Activation Energy” Potential net usable energy Now Potential net usable energy In Transition

Need to INPUT ENERGY to Destabilize Existing State Regain Activation Energy Invested net usable energy released Potential net usable energy In Transition After

What does activation energy represent? For a Reaction to Occur… - reactants must find each other, - meet in proper orientation - and hit with sufficient force

Productive Collision Many Non-productive Collisions

Needs of Typical chemical reactions - need large number of molecular collisions - need collide violently enough to break pre-existing bonds (not bounce) - need high concentration to find each other at significant rate HEAT !

The energy profile for an exergonic reaction A B C D Free energy Transition state A B C D EA Reactants A B ∆G < O C D Products Progress of the reaction Figure 8. 14

Temp 1 Temp 2 EA Molecules with sufficient Energy (~40%) Molecules with sufficient Energy (<5%)

ENZYMES make reactions easier to occur at reasonable temperature by LOWERING the ACTIVATION ENERGY EA of the reaction

Activation Energy necessary to overcome the status quo EA G “ease” of initiating reaction Thermodynamic “favorablility”

CATALYSTS: promote a specific reaction But are NOT consumed in the process Key concepts: Promotes - does not alter what would normally occur thermodynamically Specificity - promotes only one reaction, only between specific reactants to give specific products Reusable - regenerated in the process

ENZYMES are biological CATALYSTS - usually PROTEINS - sometimes RNA or RNA/protein complexes

Enzymes work as catalysts by providing an easy path to the same point Hard path Easy path HOW?

How do Enzymes do it? 1. Enzymes have BINDING AFFINITY for their reactants = Substrates Brings substrates in close proximity: conc

Stabilized Interactions Have a very Specific 3 -D Shape Charged Nonpolar Polar With a Specific Arrangement of Functional Groups Flexible OH HO HO + Enzymes act as a Specific Platform

ENZYMES: Bind ONLY specific things HO OH Bind them ONLY in a Specific 3 -D Orientation OH OH HO - HO + SPECIFICITY is the Key to Enzyme Action

2. Enzymes ORIENT Substrates always in productive orientation

Productive Collision Many Non-productive Collisions ONLY Productive Collisions

With just a little nudge, can’t help but react HO OH OH OH HO - + HO

3. Enzymes cause BOND STRAIN - destabilize existing bonds “nutcracker effect” 3 a. Physical Strain 3 b. Chemical Strain

The active site – Is the region on the enzyme where the substrate binds Substate Active site Enzyme Figure 8. 16 (a)

Induced fit of a substrate Enzyme- substrate complex Figure 8. 16 (b)

Enzyme-substrate interactions Fischer: Lock & key Koshland: Induced fit 3 a. Physical bond strain Draw an quarter - an anvil

• The catalytic cycle of an enzyme 1 Substrates enter active site; enzyme changes shape so its active site embraces the substrates (induced fit). Substrates Enzyme-substrate complex 6 Active site Is available for two new substrate Mole. Enzyme 5 Products are Released. Figure 8. 17 Products 2 Substrates held in active site by weak interactions, such as hydrogen bonds and ionic bonds. 3 Active site (and R groups of its amino acids) can lower EA and speed up a reaction by • acting as a template for substrate orientation, • stressing the substrates and stabilizing the transition state, • providing a favorable microenvironment, • participating directly in the catalytic reaction. 4 Substrates are Converted into Products.

3 b. Chemical Bond Strain tease the bond to fall apart

Chemical Bond Strain Stabilize a Fictitious state

Cofactors Non-polypeptide things at the active site that help enzymes do their job • Cofactors – Are nonprotein enzyme helpers, eg Zn++ • Coenzymes – Are organic cofactors

4. Enzymes “partake” in reactions but are not consumed in them Converts MANY “A’s” into “B’s”

H+ H+ OH- Partakes: but start and end with the same enzyme config

Lysozyme

Lysozyme: kills bacteria Works at p. H 4 -5 Why?

SUMMARY Enzymes: 1. Bring reactants (substrates) in close proximity 2. Align substrates in proper orientation 3. Can act as a Lever: a press or an anvil small shape change translates to large force 4. Release products when reaction done rebind more substrates 5. Many small steps, steps each easily achieved rather than one huge leap

Enzymes carry out reactions in a series of small steps rather than one energetic event No Problem Dude You expect me to JUMP this?

Reaction rates: Example: H 2 O 2 -> H 2 O +O 2 uncatalyzed –months Fe+++ 30, 000 x faster Catalase 100, 000 x faster Enzyme kinetics- kinetikos – moving

An enzyme catalyzed rxn Can be “saturated” Vmax maximum velocity Rate or velocity 1/2 Vmax # made per min Km Substrate Conc “substrate affinity”

The lower the Km the better the enzyme recognizes substrate “finds it at low conc” “mpg” The higher the Vmax the more substrate an enzyme can process per min (if substrate around) “top speed”

Things that affect protein structure often affect enzyme activity temperature 0 20 40 60 80 100 ºC p. H 0 1 2 3 4 5 p. H 6 7 8 9 10

Enzyme regulation: Activity controlled Continually adjusted

Principal Ways of Regulating Enzymes Competitive Inhibition Allosteric Inhibition Covalent Modification (phosphorylation)

Competitive Inhibitors: bind to active site “unproductively” and block true substrates’ access HO S 2 S 1 OH OH - I HO OH OH HO S & I bind to same site HO +

Competitive inhibition

Allosteric Inhibitors “other” “site” Distorts the conformation of the enzyme Negative allosteric regulator

Allosteric inhibition

Positive allosteric regulators Helps enzyme work better promotes/stabilizes an “active” conformation

Allosteric activation

Allosteric regulators change the shape conformation of the enzyme Allosteric enyzme with four subunits Regulatory site (one of four) Active site (one of four) Activator Active form Stabilized active form Oscillation Allosteric activater stabilizes active form Non. Inactive form Inhibitor functional active site Figure 8. 20 Allosteric activater stabilizes active from Stabilized inactive form (a) Allosteric activators and inhibitors. In the cell, activators and inhibitors dissociate when at low concentrations. The enzyme can then oscillate again.

A frequent regulatory modification Phosphorylation of enzymes

Phosphorylase kinase inactive + P active

Summary 1. enzymes are catalysts 2. Lower activation energy EA 3. Mechanism of action … 4. Enzyme kinetics- Vmax, Km 5. Regulation of enzyme activity - competitive, allosteric phosphorylation