Chapter 6: Enzyme Catalysis

Enzyme use a core set of

Catalytic strategies

Binding energy is 

The free energy released in the formation of a large number of weak interactions between the enzyme adn the substrate

What is the purpose of bidning energy?

1. Established substrate specificity

2. Increases catalytic efficiency

What is induced fit?

Process by which energy can also promote structural changes in both the enzyme and the substrate that facilitate catalysis

What is the 1st part of enzymes using 4 basic strategies for catalysis?

1. Covalent catalysis

What is covalent catalysis?

The active site contains a reactive group that becomes temproatily covalently attached to a part of the substrate

What is an example of covalent catalysis?

The proteolytic enzyme chymotrypsin

What is general acid-base catalysis?

A molecule other than water acts as a proton donor or acepetor

What is part 2?

1. Catalysis by approximation

2. Metal ion catalysis

What is catalysis by approximation?

The enzyme brings two substrates together along a single binding surface in a manner that enhances the reaction rate

What is metal ion catalysis?

Metal ions function catalytically, such as by direct coordination, stabilizing negative charged on reaction intermediates, or serving as a bridge between enzyme and substrate

Proteases facilitate

A difficult reaction

Proteases are enzymes that

Cleave proteins by hydrolysis

Proteases are thermodynamically

Favorable

Proteases kinetics are

Extremely slow

The chemical nature of peptide bonds is responsible for

Their kinetic stability

The resonance structure of the peptide bond accounts for its

Resistance to hydrolysis

The carbonyl carbon involved in the peptide is 

Less electrophilic and less susceptible to nucleophilic attack than the carbonyl carbons in more reactive compounds

Chymotrypsin posses a highly

Reactive serine residue

Chymotrypsin is a

A proteolytic enzyme that cleaves peptide bonds selectively on the carboxyl terminal side of large hydrophobic residues

Chymotrypsin uses a serine to

Attack the unreactive carbonyl carbon atom of the substrate

-becomes covalently attached to the substrate transiently

Chymotrypsin can be Inactivated:
Experimental Evidence

Diisopropylphosphofluoridate (DIPF) modifies serine 195
of chymotrypsin in a chemical modification reaction.
– chymotrypsin irreversibly loses all activity
– suggests serine 195 plays a central catalytic role

Phosphorus based agents that modify reactive serine residues can be

Potent toxins

Sarin is a 

Nerve agent that can be developed as a chemical weapon

Catalysis by chymotrypsin occurs in 2 stages:

1. A rapid step (pre-steady state)

2. Slower step (steady state)

Hydrolysis by chymotrypsin involved

Covalent catalysis

The two phases are explained by 

The formation of a covalent enzyme-substrate intermediate

What is produced as the acyl-enzyme intermediate is formed?

One molecule of p-nitrophenolate

Both phases are required for

Enzyme turnover

Serine is a part of 

A catalytic triad that also includes histidine and aspartate

Describe chymotripsyn

Roughly Spherical and contains 3 polypeptide chains linked by disulfide bonds

What marks the active site of chymotrpysin?

Serine 195

What is catalytic triad?

Set of 3 amino acid residues in the active site of some enzymes

In chymotrpysin: 

the side chain of Ser 195 is hydrogen
bonded to the imidazole ring of His 57.
– the –NH group of the His 57 imidazole
ring is hydrogen bonded to the
carboxylate group of Asp 102

Role of His 57

Positions the Ser 195 side chain and polarizes serines hydroxyl group

In the presence of substrate. His 57

acts as a general base catalyst

• Removes a proton from Ser 195

• Generates a highly reactive alkoxide ion

Asp 102 orients 

Hi 57 and renders it a better proton acceptor through hydrogen bonding and electrostatic effects

The oxyanion hole

Stabilizes the tetrahedral intermediates

What is a oxyanion hole?

A region of the active site that stabilizes:

• unstable tetrahedral reaction intermediates

• The transition state that precedes the formation of the tetrahedral intermediate

The specificity pocket of chymotrypsin favors

The binding of residues with long hydrophobic side chains

S1 pocket

Deep hydrophobic pocket into which long, uncharged side chains can fit

In S1 pocket, s ide cha in binding positions the

Adjacent peptide bond for cleavage

Catalytic triads are found

In other hydrolytic enzymes

What are homologs of chymotrypsin with different substrate specificty?

Trypsin and elastase

Differences are due to

Small structural differences in the S1 pockets

Other proteases that are not homologs of chymotrypsin contain

Similar active sites because these sites evolve independently 

Some proteases cleave peptides at other locations besides

Serine residues

What is cysteine proteases?

Rely on a Cys residue, activated by a His residue, to play the role of the nucleuphile that attacks the peptide bond

Aspartyl proteases

Use a pair of Asp residues that act together to allow a water molecule to attack the peptide bond

Metalloprotease

Use a bound metal ion (typically zinc) that activated a water molecule to act as a nucleophile to attack the peptide carbonyl group

In each of these 3 classes of enzymes, the active site includes feature that act to:

• Activates a water molecules or another nucleophile

• Polarize the peptide carbonyl group

• Stabilize a tetrahedral intermediate

Protease inhibitors are 

Important drugs

Protease inhibitors

Drugs that block protease activity

Captopril

Inhibitors of the metalloprotease angiotensin converting enzyme (ACE)

What is catpopril used for?

Used to regulate blood pressure

What is Indinavir (Crixivan)

Inhibitor of HIV protease, an aspartyl protease that cleaves multidomain viral proteins into their active forms

What does indinavir do?

Reduce deaths due to AIDS

• designed to resemble a peptide substrate of the enzyme

Carbonic anhydrases make

A faast reaction faster

What is an end product of aerobic metabolism?

Carbon dioxide

What does carbonic anhydrases convert?

Converts CO2 into bicarbonate ioni and a proton in red blood cells

Carbonic anhydrase

Dehydrate bicarbonate ion in the blood to form CO2 for exhalation

Carbonic anhydrase contains a 

Bound zinc ion essential for catalytic activity

The human genome contains

At lease seven carbonic anhydrases with its own gene

• all are homologous with susbtantial sequence identity

Zn2+ is bound to

4 ligands

Zn2+ is bound to 4 ligands

• imidazole rings of three His residues

• water molecule (or a hydroxide ion (depending on pH)

• Picture: carbonic anhydrase II

Catalysis involves

Zinc activation of a water molecule

At pH 8, 

The reaction proceeds near its maximal rate

As the pH decreases,

The rate of the reaction drops

• Midpoint is near pH 7, suggesting a group loses a proton at pH 7

Upon binding to zinc in carbonic anhydrase,

The pKa of water decreases

Zn2+ lowers the pKa of water from

15.7 to 7

At lower pKa, water can

more easily lose a proton at neutral pH to generate the potent nucleophilic OH-

Carbonic anhydrase has a 

CO2 binding site adjacent to the active site zinc ion

What serves as a binding site for CO2

A hydrophobic patch

Binding to zinc in carbonic anhydrase

The pKa of water decreases

The binding of water to the zinc ion favors

The formation of the transition state by:

• Facilitating proton release

• Positioning the water molecule to be in close proximity to the other reactant 

The rate of CO2 hydration by Carbonic anhydrase increases with..

Buffer concentration

What is a porton shuttle for large buffers

Molecular components of amny buffers are too large to reach the active site of carbonic anhydrase

What is a proton shuttle?

Transfers protons from the zinc bound water molecule to the protein surface and then to the buffer

Molecular motor proteins harness..

Changes in enzyme conformaiton to couple ATP hydrolysis to mechanical work

What is myosins?

Molecular motor proteins

What does myosins do?

• Catalyze the hydrolysis of ATP to form ADP and inorganic phosphate (Pi)

• Use the energy from this thermodynamically favorable reaction to drive molecular motion

What is the structure of myosin?

Have elongated structures

Describe myosin

• globular ATPase domains that carry
  out ATP hydrolysis at one end

• extended α-helical structures that
  promote dimer formation

• ancillary associate proteins termed
  light chains

ATP hydrolysis proceeds by the

Attack of water on the gamma phosphoryl group

The ATPase domain contains

A water-filled pocket toward the center of the structure

• ATP binding minimally changes the structure

Myosin activity requires 

Mg2+ or Mn2+

Myosins are inactive in the absense of..

Divalent metal ions bonded to ATP

Most nucleotide triphosphate (NTP) dependent enzymes require

Mg2+ or Mn2+ complexes of NTPs

What is the formation of the transition state for ATP hydrolysis

ATP hydrolysis by myosin includes a pentacoordinate transition state that is too unstable to observe 

Myosin ATPase transition state analog

Study of a more stable vanadium-based substrate
analog established the existence of a pentacoordinate
transition state.

Water attack is facilitates by

Ser 236

A water molecule attacks the 

y phosphate of ATP

The hydroxyl group of Ser 236 facilitates

The transfer of a proton from the attacking water to its hydroxyl group

Ser 236 is deprotonated by

One of the oxygen atoms of the y-phosphoryl group

A substantial conformation change is associated with

The formation of the myosin transition state

Small changes at the active site are amplified by

Large changes in the carboxyl terminal region of myosin

An actin filament has a

Polymeric structrue

Actin forms filaments along which

Myosin can move

Each actin monomer contains

A bound nucleotide, ATP, ADP

How can myosin be localized?

By being labeled with fluorescent tags