Enzymes and heart attacks

2009-2010

Enzymes:

“Helper” Protein molecules

Flow of energy through life

Life is built on chemical reactions

Chemical reactions of life

Processes of life• building molecules

• synthesis

• breaking down molecules

• digestion

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Nothing works without enzymes!

How important are enzymes?• all chemical reactions in living organisms require

enzymes to work

• building molecules

• synthesis enzymes

• breaking down molecules

• digestive enzymes

• enzymes speed up reactions

• “catalysts”

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enzyme

enzymeWe can’t live without enzymes!

Examples

synthesis

digestion

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enzyme

enzyme

Enzymes are proteins

Each enzyme is the specific helper to a specific reaction• each enzyme needs to be the right shape for the job

• enzymes are named for the reaction they help

• sucrase breaks down sucrose

• proteases breakdown proteins

• lipases breakdown lipids

• DNA polymerase builds DNA

Oh, I get it!They end in -ase

Enzymes aren’t used up

Enzymes are not changed by the reaction• used only temporarily• re-used again for the same reaction with other molecules• very little enzyme needed to help in many reactions

enzyme

substrate product

active site

It’s shape that matters!

Lock & Key model• shape of protein allows

enzyme & substrate to fit

• specific enzyme for each specific reaction

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Enzyme vocabulary

Enzyme• helper protein molecule

Substrate• molecule that enzymes work on

Products• what the enzyme helps produce from the reaction

Active site• part of enzyme

that substrate molecule fits into

What affects enzyme action

Correct protein structure• correct order of amino acids

• why? enzyme has to be right shape

Temperature• why? enzyme has to be right shape

pH (acids & bases)• why? enzyme has to be right shape

Order of amino acids

Wrong order = wrong shape = can’t do its job!

DNA

DNA

chain ofamino acids

chain ofamino acids

foldedprotein

foldedprotein

right shape!

wrong shape!

Temperature

Effect on rates of enzyme activity• Optimum temperature

• greatest number of collisions between enzyme & substrate

• human enzymes • 35°- 40°C (body temp = 37°C)

• Raise temperature (boiling)

• denature protein = unfold = lose shape• Lower temperature T°

• molecules move slower • fewer collisions between enzyme & substrate

37°

Temperature

temperature

reac

tion

rat

eWhat’s happening here?!

humanenzymes

How do cold-blooded creatures do it?

pH Effect on rates of enzyme activity• changes in pH changes protein shape

• most human enzymes = pH 6-8

• depends on where in body

• pepsin (stomach) = pH 3

• trypsin (small intestines) = pH 8

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pH

pH

reac

tion

rat

e

20 1 3 4 5 6 8 9 10

stomachpepsin

intestinestrypsin

What’s happening here?!

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2009-2010

For enzymes…What matters?

SHAPE!

2009-2010

Let’s build some

Enzyme Models!

Myocardial infarction

Acute myocardial infarction is the rapid development of myocardial necrosis caused by a critical imbalance between the oxygen supply and demand of the myocardium.

500,000-700,000 deaths inthe US annually.

Myocardial infarction

Symptoms• Angina pectoralis

• Dyspnea

• Nausea and/or abdominal pain

• Anxiety

• Lightheadedness and syncope

• Cough

• Nausea and vomiting

• Diaphoresis

One problem - Differential diagnosis• Pericarditis • Aortic Dissection • Cholecystitis and

Cholelithiasis • Laryngeal spasm• Anxiety attack• and on and on and on…

One solution – “Cardiac enzymes”

Enzymes

Definition: Biological catalysis

Qualities• Efficient

• Specific

• Stereo-specific - they can tell the difference between isomers

• Regulated

• Saturable

• Inhibitable

Substrate versus product

)(sproductsubstrate enzyme

2222 223

OOHOHFe

catalase

Types of enzymes

All enzymes end in the suffix “_______ase”

Different versions of the same enzyme (often made by alternative splicing) are called isoenzymes or isozymes

General classes of enzymes • Polymerases – nucleic acid synthesis• Transferases – transfer a functional group• Hydrolases – hydrolytic cleavage• Proteases – hydrolytic cleavage of protein chains• Kinases – add phosphate groups to compounds• … and many, many more…

Mechanism

Enzymes work by lowering activation energy• If you don’t understand free energy

changes, see Box 5A in your book

∆G is a measure of the ability of a reaction to go forward, but not necessarily the rate

EA is the activation energy.

The rate at which a reaction proceeds is directly proportional to the number of molecules reaching the transition state - that is, those that reach EA. 

Things for optimal activity

pH – alters enzyme structure by altering charge

Temperature – increases activity by moving molecules closer to the activation energy, and by making ∆G slightly more negative… until the enzyme "denatures"

Coenzymes – like biotin in amino group transfer – bind reversibly but participate directly

Metal ions – like magnesium in some ATPases.

Michaelis-Menten Kinetics

Shows saturation at high substrate concentrations

Vmax – rate at saturation for a given enzyme concentration in moles per unit time

Km – Michaelis constant – substrate concentration that gives ½ maximal velocity

SK

SVV

m max

How do you measure this crap?

Things you need:• The enzyme• The substrate• A way of

measuring either the disappearance of substrate, or the appearance of product, usually photometrically.

Other commonly reported values

Turnover • rate at saturation for 1 enzyme molecule

(reactions catalyzed per second per molecule)

“Units” • are defined by convention, but are something of

an industry standard.  For example…• “One unit of creatine kinase is defined as the

amount necessary to catalyze the conversion of one micromole of creatine to creatine phosphate per minute at 25°C and pH 8.9.”

Competitive inhibitors

Many drugs (like Cipro and anti-HIV drugs) are enzyme inhibitorsTwo major kinds of inhibitors: competitive and noncompetitive.Competitive inhibitors bind to the active site of the enzyme.

Alter Km but not Vmax.

What will happen to V ifyou push the substrateconcentration very high?

Noncompetitive inhibitors

Noncompetitive inhibitors bind somewhere besides the active site.

They alter the behavior of the enzyme in a manner analogous to allosteric regulation

Alter Vmax.

What will happen to V ifyou push the substrateconcentration very high?

RegulationAllosteric regulation

A regulatory molecule binds to a site separate from the active site (like small molecules to repressors in operons)Induced conformational changes regulate the activity of the enzymeThese enzymes usually have catalytic and regulatorydomainsCan have multiple domainsor subunits for different regulators

Regulation

AllostericCooperativity

• One substrate aids or impedes the catalysis of another• Implies multiple catalytic subunits.

Covalent modification• Adding/removing groups – like phosphate groups by

kinases • Cleaving bonds – converting proenzymes to enzymes -

like in the blood clotting cascade

Association-dissociation of subunits• One protein binds to another, thereby activating the

enzymatic activity of one of them.

Creatine kinase

Creatine phosphate acts as a backup for rapid ATP regeneration in active tissues• Creatine phosphate is in energetic

equilibrium with ATP

• Creatine kinase (CK) catalyzes the transfer of phosphate between creatine and ATP/ADP

Provides rapid regeneration of ATP when ATP is low

Creatine phosphate is regenerated when ATP is abundant

CrCrCr-PCr-P

ATPATPADPADP

CK

Application: Cardiac enzymes

enzymes released from injured myocardium.

Creatine kinase (CK) is the one usually assayed

If CK is found in the blood stream, this implies that the myocardium may have been damaged

Problems:• Tells you little about the time course or severity

• Lets you spot really small infarcts.

• What else?

Creatine kinase isozymesThe enzyme is dimeric

Two different polypeptide chains (M and B) are differentially expressed in tissues

Combine at random to give three isozymes: • CK-MM (primarily muscle)

• CK-MB (hybrid)

• CK-BB (primarily brain)

The CK-MB has its highest concentration in heart muscle

CK-MB >5% of total CPK strongly suggests myocardial infarction

Total CK activity is determined by a simple enzyme assay (phosphocreatine + ADP ATP)

CK-MB mass is determined by a two-antibody “sandwich” assay.

Determining CK-MB (mass) / CK (activity)

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anti-CK-B coated tube

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Tagged anti-CK-M

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Y

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Y

POSITIVE

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Y

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Y

Y

Y

Substrate