Introduction to Enzymes

Lecture 14. 04/23/2015

Reading: Lehninger 6.1 - 6.2Voet, Voet, and Pratt pg. 315-323; 331-332

Key terms and topics:Enzymes

OxidoreductaseTransferaseIsomeraseHydrolaseLyaseLigase

Co-factorsCo-enzymesSubstrateCatalysisActive site and models for fitActivation energyBinding energyTransition state

PepsinPDB: 5PEP

What is Catalysis and What is an Enzyme?

Sucrose CO2 + H2O is very exergonic (energetically favorable)

Historical Background

Late 1700s early 1800s

1850’s Louis Pasteur

1897 Eduard Buchner

1926 James Sumner

Meat digestion by stomach secretions observed. Saliva and some plant extracts can convert starch to sugar.

Postulated that the fermentation of sugar into alcohol is done by “ferments”, inseparable from the cell.

Yeast extracts can also ferment sugar.

Crystallized Urease

Background and Nomenclature

Background and Nomenclature

• Enzymes often have other stuff in themCofactors—inorganic ionsCoenzymes—complex organic or metalloorganic molecules

transient carriers of specific functional groupspermanently bound coenzymes are called prosthetic groups

Cytochrome

Chlorophyll

Vitamin B12

FAD

IP6

Features of an Enzyme Enzymes are macromolecules

Catalyze (increase the rate) the conversion of substrate to product

Enzymes are not changed by the reaction

Catalysis occurs in the ‘active site’ of the enzyme

Most enzymes are large (>100 aa’s)

Enzymes are regulated

Background and NomenclatureEnzymes can be named for their function and/or substrate and often end with –ase.

Useful but sometimes ambiguous

Enzymes can be named systematically by their Enzyme Commission Number (E.C.)

Uses substrate(s) or product(s) and enzyme class

ex. ATP + β-D-glucose 6-phosphate ADP + β-D glucose 1,6-bisphosphate

H3C CH

OH

COOH H3C C

O

COOH + NADH + H+

PyruvateLactate

+ NAD+

Common Name: Lactate Dehydrogenase

Systematic name:

Reverse: NADH:pyruvate Oxidoreductase

Oxidoreductase: transfer of e-

(oxidation-reduction reactions)

Common Name: PhosphofructokinaseSystematic name:

Reverse: Not named (except for kinases)

+ ADPH3C CH

OH

COOH H3C C

O

COOH + NADH + H+

PyruvateLactate

+ NAD++ ATP

Fructose-6-phosphate Fructose-1,6-bisphosphate

Transferase: transfer of functional groups(must be between two molecules)

Common Name: Triose phosphate isomerase

Systematic name:

Reverse: Glyceraldehyde-3-Phosphate isomerase

H3C CH

OH

COOH H3C C

O

COOH + NADH + H+

PyruvateLactate

+ NAD+

Dihydroxyacetonephosphate Glyceraldehyde-3-phosphate

Isomerase: intramolecular rearrangement(must be within same molecule)

Protease

Systematic name:

Always named in this direction

H3C CH

OH

COOH H3C C

O

COOH + NADH + H+

PyruvateLactate

+ NAD++

H2O

H2O

Hydrolase: single bond cleavage via addition of H2OOr bond formation via the removal of H2O

Common Name: Enolase

Systematic name:

Always named in this direction

H2O

Substrate(s) Product(s)

H2O

2-phosphoglycerate Phosphoenolpyruvate

Lyase: group elimination to form a double bond(can be a functional group or part of the molecule

Common Name: Pyruvate Carboxylase

Systematic name:

OR: Oxaloacetate synthetase

Substrate(s) Product(s)+

ATPADP +

Pi

ATP ADP + Pi

Pyrvuate oxaloacetate

Ligase: bond formation coupled to ATP hydrolysis

A. Oxidoreductase

B. Transferase

C. Ligase

D. Lyase

E. Hydrolase

What class does the following enzyme belong

to?

The Active Site of an Enzyme

Active siteSubstrate

Lock and Key Induced Fit

Models for Ligand Binding to Active Site

How do Enzymes Increase the Rate of the Reaction?It has a lot to do with free energy, G

E + S ES EP E + P

Reaction coordinate diagrams plot the free energy change as the reaction proceeds.

The free energy of the product is lower than the

free energy of substrate in their ground (or intital)

states.

G’° is negative and the equilibrium favors P.

It says nothing about how fast the reaction

occurs. It may take years.

Reaction Coordinate Diagram for Enzyme Catalyzed Reaction

Enzyme lowers the energy required to reach transition state, they reduce the energy barrier between S and P.

E + S ES EP E + P↔ ↔↔

How do Enzymes Lower the Activation Energy?

This requires energy - where does it come from?

Enzymes can increase the rate of a reaction anywhere from 5 to 17 orders of magnitude (that’s 105 to 1017

times) by lowering the activation energy.

Binding Energy Helps Drive Catalysis

Weak Interactions are Optimal Between the Enzyme and the Transition State

Transition State Theory Relates the G (the change of G) of Uncatalyzed and Catalyzed Reactions to the Reaction Rate

X‡ = transition state

S X‡ ES EX‡

It explains how a relatively small decrease in activation energy translates to a huge increase in rate!

K =[X‡][S]

Kenz =[EX‡][ES]

uncatalyzed rxn catalyzed rxn

[X‡] = K [S] [EX‡] = Kenz [ES]

(assume all enzyme bound by S so [ES] = [S])

G = -RTlnK

lnKeq = -G/(RT)

K = e -G/(RT)

If the enzymes stabilizes the transition state by 5.7 kJ/mol(meaning Gu - Genz = G = 5.7 kJ/mol)

what is the rate enhancement with the enzyme?

A Small Change in G in Activation Energy Between the Uncatalyzedand Catalyzed Reaction Results in Huge Changes in Rate

The proof?

Clicker Question

Which of the following is true of the transition state?

A. It represents the enzyme-substrate (ES) complex.

B. ΔG of the reactants going to the transition state is negative (favorable).

C. Amino acids are precisely arranged to specifically bind the transition state.

D. It increases the KD between substrate and enzyme.

E. All of the above are true.