Introduction to Enzymes

Biological Catalysts

Life Process = Chemical Reactions

Enzyme Catalysis
Substrate(s)
in theory, reversible

Product(s)
depending on which one is favorable, can do both directions

Life Process = Chemical Reactions

product of one reaction becomes a substrate for another reaction individual enzyme that catalyze the reaction and overall characteristics/regulation of pathway

Enzymes
A
Enzyme 1

Enzyme 2

Enzyme 3

Enzyme 4

Enzyme 5

PATHWAY

Chemical Reactions
speed up reactions cannot determine spontaneity aka if reaction happens thermodynamically favorable - spontaneous

Spontaneous and Fast

thermodynamically AND kinetically favorable

HCl + NaOH

Na + Cl + H2 O
must add catalyst thermodynamically favorable, but won't happen fast

Spontaneous but Slow

2 H2 + O2 ATP + H 2O

2 H2 O ADP + Pi

Types of Reactions
Spontaneous Reactions:
thermodynamically or energetically favorable Kinetically Unfavorable Reactions
no catalyst? reaction does not proceed fast enough to support life - have a -deltaG in the cell, does not mean standard reactions are thermodynamically favorable

Requirement for Catalysts

Protein Catalysts: Enzymes
RNA Catalysts: Ribozymes

vast majority of catalysts are proteins

General Properties of Enzymes

Higher reaction rates (catalytic power) Milder reaction conditions Greater reaction specificity Capacity for regulation
can determine when they function speed up reactions

advantages

Higher Reaction Rates

Carbonic Anhydrase

CO2 + H2O

H2 CO3

105 molecules CO2 per enzyme molecule per second 107 x uncatalyzed reaction

Catalytic Power of Some Enzymes

range of rate enhancements more reactions per second

Table 11-1

Mild Reaction Condition Physiological pH = ~7.4 Temperature = ~37C

no side reactions; only a specic reaction can have enzymes that catalyze a single reaction or use multiple substrates for a single reaction allows for diversity and exibility in reactions can regulate/control the reactions

Greater Reaction Specificity

Hexokinase

Glucose + ATP Fructose + ATP Mannose + ATP

Glucose-6-P + ADP Fructose-6-P + ADP Mannose-6-P + ADP

Glucokinase Glucose + ATP ONLY Glucose-6-P + ADP

Table 11-1

Capacity for Regulation

two categories on how we regulate enzyme activity

Covalent Modification

physically changing the enzyme

Irreversible cleaving off a part of peptide Reversible ex. phosphorylation/dephosphorylation - changes activity Non-covalent Modification
Allosteric (Regulatory) Enzymes
small molecules that bind to an enzyme + affect activity can be released

Enzyme Classes
Oxidoreductases
Transferases

Isomerases
Hydrolases Lyases Ligases

(Usual usage: often use suffix ase)

Enzyme Nomenclature

Common Name:
Useful but sometimes ambiguous
Examples: Urease/Arginase Exceptions to the ase suffix: Trypsin/Chymotrypsin
generally use common name

Systematic Name:
Substrate(s) Type of reaction-ase
specic substrate + specic reaction

oxidation-reduction reactions (transfer of electrons)

one that loses electrons, one that gains

Oxidoreductase:

OH H3C CH COOH + NAD+

cofactor will be reduced

O H3C C COOH + NADH + H+

Lactate
substrate that is oxidixed

Pyruvate
substrate will lose hydrogens

hydride
transferring electrons not protons

Common Name: Lactate Dehydrogenase Systematic name: L-Lactate:NAD+ Oxidoreductase

e-donor e-acceptor

transfer of functional groups

new functional group

Transferase:

OH H3C CH COOH +ATP + + NAD H3C

O C COOH + NADH + + ADP H+

Lactate

Pyruvate

Common Name: Phosphofructokinase Systematic name: Fructose-6-phosphate kinase

Phosphate transferase = kinase

substrate, functional group being transferred, type of molecule

intramolecular rearrangement
apparent intramolecular rearrangement - does not have to be the same functional group that is being trasferred

Isomerase:

OH H3C CH COOH + NAD+ H3C

O C COOH + NADH +

Lactate

Pyruvate

Common Name: Triose phosphate isomerase

Systematic name: DihydroxyacetonePhosphate isomerase
substrate + isomerase

Single bond cleavage via addition of H2O

H2O
OH H3C CH COOH + NAD+ H3C

Hydrolase:

O C COOH + NADH + H+

Lactate

H2O

Pyruvate

Systematic name: Compound to be cleaved + hydrolase

peptide hydrolase

Group elimination to form a double bond

not all lyase has a water that can be removed important - double bond not cleaving a molecule

Lyase:

H2O

Substrate(s)
H2O

Product(s)

Common Name: Enolase

Systematic name: 2-phosphoglycerate Lyase OR: Phosphoenolpyruvate synthase
or product name w/ double bond + synthase
substrate w/o double bond + lyase

bond formation coupled to ATP hydrolysis

ligase - synthetase liase - synthases add CO2 group

Ligase:

new single bond + hydrolysis of ATP

ATP

ADP + Pi

Substrate(s) +
ligase - complete ATP hydrolysis 1 side ATP - 1 side ADP + Pi cleaved, not transferred when it is not interacting with anything, it is ligase

Product(s)
ATP ADP + Pi

Common Name: Pyruvate Carboxylase

OR: Oxaloacetate synthetase
products (single molecule) + synthetase two molecules being joined together + ligase

Systematic name: Pyruvate Carbon Dioxide ligase

Reaction Pathway (Coordinate)

stabilizing transition state in a mechanism

CH3Br

+ OH

CH3OH

Br

H OH
-

H C Br HO C H

H Br HO C

H H H + Br
-

+ H
H

Reactants

"Transition State"
all ve groups are bonded at once generally unfavorable/high energy stabilized by enzyme

Products

Transition State Diagram

high energy intermediate

determines how fast reaction occurs

Stabilizing the transition state

does not happen on its own

if it binds to substrate really well, will bind to substrate but nothing else happens

would increase energy of activation would not catalyze reaction

binds transition state better or as good as substrate has some features of substrate but is not as comfortable more likely to get transition state to occur helps make it lower in energy state

Catalysts

binds preferentially to TS, and lowers activation energy

Pathway of Enzyme Catalysis

Binding
two steps - one will be rate limiting, depending on enzyme

E+S

[ES]
Complex

Catalysis

E+P

EnzymeSubstrate
one will be limiting

Active Site
where binding occurs

Substrate Specificity
Active Site
determines specicity organized so that we have both stereospecicity and geometric

Stereospecificity:
3-point attachment

demand that a single isomer is generated

Geometric Specificity:

exclude some substrate

e.g. trypsin and chymotrypsin

Through Complementarity

both are generated through complementarity

Models of Complementarity
way that active site binds to ligand or substrate

Lock and Key

active sites are in a static specic orientation only exact orientation will bind

Induced Fit
active site is more dynamic exact geometry of ligand will change active site so it will be complementary to ligand

depends on how many substrates will be used

if there is only one substrate probably lock and key if it can catalyze similar reactions will multiple substrates induced t

Principle of Complementarity

Geometric (physical) complementarity

Electronic (chemical) complementarity
right size to t into substrate and right chemical characteristics (hydrophobic vs polar vs charged)

Enzyme-Substrate Complex

Binding Site

Enzymes are Stereospecific

Aconitase Reaction
in TCA cycle

will only form the R isocitrate

active site is arranged that only pro R is available in active site

Prochiral Substrate
not chiral has two identical arms, modify one will be chiral
Page 325

Chiral Product

Stereospecificity in Substrate Binding

active site can bind three of them
only one arm that will get something added the pro R arm

Figure 11-2

Enzymes Vary in Geometric Specificity

(Alcohol Dehydrogenase)
Ethanol > Acetaldehyde
altering size of active site alcohol dehydrogenase can use all these as substrates

Methanol > Formaldehyde

Isopropanol > Dimethylketone

there is a hierarchy to which it forms

RATE: Ethanol > Methanol > Isopropanol

size of active site alcohol dehydrogenase will t ethanol just ne not as great for methanol or isopropanol

Trypsin and Chymotrypsin

both cleave peptide bonds but have specicity to what peptide bond they cleave after

H2O O NH CH R1 C NH CH R2 O C

O NH CH R1 C

_ O

+ H 3N

O CH R2 C

Trypsin
H2O
O N NH CH C NH

amino acid binding site is longer cleaves after long positively charged side chain

arginine or lysine

Long + side chain"charged "long positively side chain

complementary binding or positioning site

has negative charge to combat positive

"SPECIFICITY"

bulkier, hydrophobic pocket

Chymotrypsin
H2O
O N NH CH O C NH C

phenylalanine tyrosine tryptophan

"aromatic side chain" Aromatic side chain complementary binding or positioning site

Hydrophobic Pocket

"SPECIFICITY"

Some Enzymes Require Cofactors

Cofactors
Simple Proteins (no cofactor)

Protein plus Cofactor

Apoenzyme: protein only inactive

Holoenzyme: protein plus cofactoractive when bound to cofactor

Apoenzyme + cofactor
(inactive)

Holoenzyme
(active)

Types of Cofactors

Organic Cofactor

Transiently Associated

Permanently Associated

Figure 11-3

Metal Ions

used by enzyme in catalysist why these trace elements are essential

cofactors for essential enzymes why heavy enzymes are toxic same column as metal ion cofactor - can bind to enzyme but can't perform catalysis ex. mercury can bind like zinc

Coenzymes: Cosubstrates
[NAD(P)+ > NAD(P)H + H+]

both in oxatodase reactions

NAD(P) - binds before or with substrate undergoes reduction then is released

catabolic reactions

two forms - phosphate group is the difference

Figure 11-4

anabolic (biosynthetic) reactions

Coenzymes: Cosubstrates
(Alcohol Dehydrogenase)

also uses as cofactor

Page 327

Coenzymes: Prosthetic Groups

(Cytochromes)

permanently associated

CytochromeHeme(Fe3+)
changing oxidation states with e-

CytochromeHeme(Fe2+)

Coenzymes Must be Regenerated

2H+ + 2e NAD+ 2H+ + 2e
whether they are permanently associated or released must be regenerated

e NADH + H+ Heme(Fe3+) e Heme(Fe2+)

Alcohol Dehydrogenase Cosubstrate: Different enzyme

released by enzyme, must be regenerated with another enzyme before reaction continues
part of enzyme - important

Cytochromes Prosthetic group: Same enzyme

because they are permanently attached, catalytic reaction will regenerate both not complete until both are in same state as they were in beginning

General Properties of Enzymes

Biological Catalysts
Not used up in the reaction (Regenerated)
Higher reaction rates (catalytic power)
Within a biologically relevant time frame

Milder reaction conditions

cannot change whether it is thermodynamically favorable

Biologically appropriate conditions

Greater reaction specificity Capacity for regulation

Control of substrate and product availability
can physically modify the activity of enzyme or control the availability of substrate/product