Enzyme catalytic strategies and regulation

Protease: example of catalytic strategies • Tetrahedral intermediate then breaks down as the amine "half" of original peptide leaves • Reason uncatalyzed reaction is so slow: partial d

Enzyme: Catalytic Strategies and

Regulation

Instructor: Dr Nguyen Thao Trang

School of Biotechnology Semester I 2015-2016

ADVANCED BIOCHEMISTRY

Acid-base catalysis

• Specific functional groups in enzyme structure positioned to:

– Donate a proton (act as a general acid), or

– Accept a proton (act as a general base)

• General acid catalysis: proton transfer from an acid

lowers the free energy of a reaction’s transition state

• General base catalysis: reaction rate is increased by

proton abstraction by a base

(a) Uncatalyzed

Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

Acid-base catalysis

• The side chains of the amino acid residues Asp, Glu,

His, Cys, Tyr, and Lys act as acid and/or base catalysts

enzyme alters pathway to get to product

• This covalent bond is formed by the reaction of a nucleophilic group on the catalyst with an electrophilic group on the substrate  nucleophilic catalysis

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Covalent catalysis

• Nucleophile: an electron-rich group that attacks nuclei

• Electrophile: an electron-deficient group

Metal ion catalysis

• Nearly 1/3 of all known enzymes require metal ions for catalytic activity

• Metal ions can be

– Tightly bound (metalloenzymes), i.e., as a prosthetic group

(usually transition metal ions, e.g., Fe2+ or Fe3+, Zn2+, Cu2+,

Mn2+…) – Loosely bound, binding reversibly and dissociating from enzyme (usually Na+, K+, Mg2+, Ca2+ )

• Functions of metal ions in catalysis:

– Binding and orientation of substrate (ionic interactions with negatively charged substrate)

– Redox reactions (e.g., Fe2+ / Fe3+ in some enzymes)

– Shielding or stabilizing negative charges on substrate or on transition state (electrophilic catalysis)

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Metal ion catalysis

• Example:

Carbonic anhydrase

Active site of carbonic anhydrase

H2O polarized by Zn 2+ ionizes to form OH which nucleophilically attacks the enzyme- bound CO2:

Reaction rate is 24 times faster

Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

Examples of catalytic strategies

• Myosin motor domain ATPase

– An enzyme that couples the hydrolysis of ATP to the mechanical motion

Protease: example of catalytic strategies

• Proteins must be degraded so that their constituent amino acids can be recycled for the synthesis of new proteins

• Protein degradation via proteolytic cleavage pathway:

• Mechanism: simple nucleophilic attack by :O of H2O on carbonyl C of peptide bond, forming tetrahedral intermediate:

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Protease: example of catalytic strategies

• Tetrahedral intermediate then breaks down as the amine

"half" of original peptide leaves

• Reason uncatalyzed reaction is so slow: partial double bond character of peptide bond makes carbonyl C much less reactive than carbonyl Cs in carboxylate esters:

• Catalytic task of proteases is to make that normally

unreactive carbonyl group more susceptible to nucleophilic attack by H2O

Protease: example of catalytic strategies

• 4 classes of proteases based on different mechanisms to enhance the susceptibility of the carbonyl group to nucleophilic attack:

1 Serine (Ser) proteases: covalent catalysis, with initial nucleophilic

attack carried out by enzyme Ser-O(H) group made into a potent nucleophile with assistance of nearby His imidazole that acts as a general base

2 Cysteine (Cys) proteases: again, covalent catalysis, with initial

nucleophilic attack carried out by an enzyme Cys-S(H) group made into a potent nucleophile with assistance of nearby His imidazole

3 Aspartic acid (Asp) proteases: nucleophile is HOH itself, assisted

and orientation/polarization of substrate by 2nd Asp

4 Metalloproteases: again, nucleophile is HOH, but assisted by

binding to a metal (e.g Zn2+) and by general base catalysis by

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Serine protease: Chymotrypsin

• Chymotrypsin participates in the breakdown of proteins

in the digestive system

• Chymotrypsin cleaves peptide bonds selectively on the carboxyl terminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine:

Biochemistry, Tymoczko, Berge, Strayer

Serine protease: Chymotrypsin

• The active site of chymotrypsin is serine 195 residue:

When treated with organofluorophosphates such as

diisopropylphosphofluoridate (DIPF), chymotrypsin lost all activity irreversibly

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Biochemistry, Tymoczko, Berge, Strayer

Serine protease: Chymotrypsin

• Catalytic mechanism of chymotrypsin:

2 half reactions or 2 phases of catalysis, with an

acyl-enzyme intermediate between the 2 half reactions

• Phase 1: Acylation

– Enzyme provides potent nucleophile, a specific Ser O(H) group Ser OH made more nucleophilic than usual with assistance of nearby His residue as general base

intermediate Amine "half" of original peptide/protein released as product (P1) at end of first phase

P1

Serine protease: Chymotrypsin

• Phase 2: Deacylation

– 2nd substrate, H2O, is nucleophile, attacking carbonyl C of the carboxylate ester of acyl enzyme, again with assistance of active site His residue as general base

– Ester bond of intermediate is hydrolyzed to regenerate alcohol component (the enzyme chymotrypsin, with its Ser-OH free

(carboxyl "half“ of original substrate peptide/protein)

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Biochemistry, Tymoczko, Berge, Strayer

P2

Serine protease: Chymotrypsin

• Serine is part of a catalytic triad

Amino acid residues in active site in a hydrogen-bonded network: – Ser (residue # 195)

– His (residue # 57)

– Asp (residue # 102)

Essential for effective catalytic activity in chymotrypsin

Catalytic triad action converts OH group of Ser 195 into a potent

nucleophile:

Serine protease: Chymotrypsin

• Complete mechanism: acid-base and covalent

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Biochemistry, Tymoczko, Berge, Strayer

Serine protease: Chymotrypsin

First phase: Acylation

• Polypeptide chain of substrate also forms a short β-sheet

(hydrogen bonds) with a β strand

of enzyme in binding site

Biochemistry, Tymoczko, Berge, Strayer

Serine protease: Chymotrypsin

– Ser-O(–) (potent nucleophile) carries out nucleophilic attack on carbonyl

C of substrate (nucleophilic catalysis, i.e covalent catalysis) >

COVALENT bond to carbonyl C (1 st tetrahedral intermediate) – Asp in catalytic triad: a) helps maintain perfect orientation of His and Ser residues in hydrogen bonded network, and b) facilitates H + transfer by electrostatic stabilization of HisH + after it has accepted the proton

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Biochemistry, Tymoczko, Berge, Strayer

Serine protease: Chymotrypsin

• Product of step 2 (nucleophilic attack in acylation half-reaction) = 1 st TETRAHEDRAL INTERMEDIATE

• There are now 4 atoms bonded to the carbonyl carbon, arranged as

a tetrahedron, instead of 3 atoms in a planar arrangement

• Tetrahedral intermediate bears a formal negative charge on the

oxygen atom derived from the carbonyl group This charge is

stabilized by interactions with NH groups from the protein in a site

termed the oxyanion hole

• Oxyanion hole is an area in the active

site of serine proteases that binds the

transition state particularly tightly

• Active site binds oxyanion more tightly

than it bound original carbonyl group of

the substrate

• An additional hydrogen bond forms

between tetrahedral oxyanion and

Serine protease: Chymotrypsin

3 Formation of acyl-enzyme intermediate

– 1st tetrahedral intermediate breaks down: original amide

(peptide) bond cleaves – HisH+ donates a proton to the amino "half" of the original

conversion of oxyanion back into a C=O, still covalently attached

to Ser residue of enzyme, forming acyl-enzyme intermediate

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Biochemistry, Tymoczko, Berge, Strayer

Serine protease: Chymotrypsin

4 Amine product (R2-NH2) dissociates from active site

(1st product leaves)

• Amine product (R2-NH2) dissociates

from the active site

• Original carbonyl group of peptide bond is now a carbonyl group

again, but it's covalently attached to

the Ser-O in the acyl-enzyme

product of first half reaction (acylation phase)

Serine protease: Chymotrypsin

• 2nd phase: Deacylation-Breakdown of acyl-enzyme

of the carboxylic acid product

5 Binding of 2nd substrate, H2O, in active site

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Biochemistry, Tymoczko, Berge, Strayer

• Nucleophilic attack facilitated by HisN:

acting as general base (but nucleophile is

H 2 O, attacking carbonyl C of acyl-enzyme)

Serine protease: Chymotrypsin

6 Formation of 2nd tetrahedral intermediate

– HOH forms hydrogen bond with HisN: in catalytic triad

– His again acts as a general base, to become HisH+, activating O from

H2O to make it a potent nucleophile, to attack carbonyl C of enzyme intermediate (an ester)

acyl-– Nucleophilic attack of HOH on carbonyl C of acyl-enzyme intermediate

→ covalent bond between OH of water and carbonyl C  2 nd tetrahedral intermediate

– Asp in catalytic triad: a) helps maintain perfect orientation of catalytic triad, and b) facilitates H+ transfer by electrostatic stabilization of HisH+ after it has accepted the proton

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Serine protease: Chymotrypsin

– HisH+ (general acid) donates proton back to Ser O, generating Ser-OH

– Ester bond from acyl-enzyme intermediate breaks > carboxylic

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Biochemistry, Tymoczko, Berge, Strayer

Serine protease: Chymotrypsin

8 Carboxylic acid product dissociates from active site

Enzyme molecule now in its original state, with His imidazole in neutral form, catalytic triad appropriately hydrogen-bonded, and active site ready to bind another molecule of substrate and do it all again

Biochemistry, Tymoczko, Berge, Strayer

The hydrophobic “specificity pocket” of chymotrypsin

• Why chymotrypsin prefers to cleave

the peptide bonds on the carboxyl

terminal side of the large hydrophobic

amino acids?

- Area of active site responsible for the

substrate specificity of chymotrypsin

- The presence of a deep hydrophobic

pocket to which the large, long

hydrophobic side chains of residues

aromatic ring bound in pocket is

shown in green in center)

- Note Gly residues in “lining” of pocket

(small, so bulky, hydrophobic side

chains fit in binding site)

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Biochemistry, Tymoczko, Berge, Strayer

Homologs of Chymotrypsin

• Many other peptide-cleaving proteins, trypsin and elastase, contain catalytic triads similar to that discovered in chymotrypsin homologs of chymotrypsin

• The sequences of these proteins are approximately 40% identical with that of chymotrypsin, and their overall structures are quite similar

• These proteins operate by mechanisms identical with that of chymotrypsin

Fig Structural similarity of trypsin and chymotrypsin

An overlay of the structure of chymotrypsin (red) on that of trypsin (blue) is shown

Biochemistry, Tymoczko, Berge, Strayer

Specificity pockets of chymotrypsin homologs

• 3 enzymes differ markedly in substrate specificity:

– Trypsin cleaves peptide bonds on carbonyl side ("after") long and charged residues (R1 = Lys + or Arg + ) Specificity is assisted by Asp–residue in bottom of the pocket

– Pocket of elastase is partly closed off so only small side chains may

enter (Val residues instead of Gly residues in the lining of the pocket)

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Fig The specificity pockets of chymotrypsin, trypsin, and elastase

Biochemistry, Tymoczko, Berge, Strayer

Protease: example of catalytic strategies

• 4 classes of proteases based on different mechanisms to enhance the susceptibility of the carbonyl group to

nucleophilic attack:

1 Serine proteases (e.g., chymotrypsin): covalent catalysis, with

initial nucleophilic attack carried out by enzyme Ser-O(H) group

made into a potent nucleophile with assistance of nearby His

imidazole that acts as a general base

2 Cys proteases: again, covalent catalysis, with initial nucleophilic

attack carried out by an enzyme Cys-S(H) group made into a

potent nucleophile with assistance of nearby His imidazole that

acts as a general base

3 Asp proteases: nucleophile is HOH itself, assisted by 2 Asp

orientation/polarization of substrate carbonyl by 2nd Asp residue

4 Metalloproteases: again, nucleophile is HOH, but assisted by

binding to a metal (e.g Zn2+) and by general base catalysis by

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Activation strategies for 3 more classes of proteases

• Purpose: activation of carbonyl C of peptide bond for

attack by a nucleophile

• All generate a potent nucleophile to attack peptide

carbonyl group

• Cysteine proteases: nucleophile is a Cys thiol activated

by His (general base)

do not require the full catalytic triad but only His

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Biochemistry, Tymoczko, Berge, Strayer

Activation strategies for 3 more classes of proteases

• Aspartyl proteases: nucleophile is HOH itself assisted

carboxyl group and orientation/polarization of substrate carbonyl by 2nd Asp residue

– 1st Asp (in its deprotonated form) attacks H2O

Biochemistry, Tymoczko, Berge, Strayer

Activation strategies for 3 more classes of proteases

• Metalloproteases: nucleophile is HOH assisted by

binding to a metal (e.g Zn2+)

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Biochemistry, Tymoczko, Berge, Strayer

Clinical insight: HIV Protease is an Asp protease

• HIV protease: cleaves multidomain viral proteins into

their active forms; blocking this process completely

prevents the virus from being infectious

• Is a homodimer: 2 identical subunits, each contributing

an Asp to active site

Biochemistry, Tymoczko, Berge, Strayer

Fig HIV protease, a dimeric aspartyl protease

2 identical subunits, shown in blue and yellow, consisting of 99 amino acids each

Notice the placement of active-site aspartic acid residues, one from each chain, which are shown as ball-and-stick structures The flaps will close down on the binding pocket after substrate has been bound

HIV Protease is an Asp protease

• Indinavir (Crixivan): Is used in the treatment of AIDS

• Indinavir resembles the peptide substrate of the HIV

protease Indinavir is constructed around an alcohol that mimics the tetrahedral intermediate

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Biochemistry, Tymoczko, Berge, Strayer

HIV Protease is an Asp protease

• Indinavir (Crixivan):

– In the active site, indinavir adopts a conformation that

approximates the twofold symmetry of the enzyme – The active site of HIV protease is covered by two flexible flaps that fold down on top of the bound inhibitor

– The OH group of the central alcohol interacts with 2 Asp

residues of the active site

- Indinavir thus inhibits HIV protease without affecting normal cellular Asp proteases, which don't have the 2-fold symmetry that HIV protease has

Examples of catalytic strategies

• Myosin motor domain ATPase

– An enzyme that couples the hydrolysis of ATP to the mechanical motion

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Carbonic anhydrase

producing) metabolic pathways

Carbonic anhydrase

• The nucleophile in this reactions is HOH

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The structure of human carbonic anhydrase II and its zinc site

Zn 2+ is bound to the imidazole

rings of 3 His residues as well as

to a H2O molecule

Biochemistry, Tymoczko, Berge, Strayer