Download ppt - Lecture 17 –Exams in Chemistry office with M’Lis. Please show your ID to her to pick up your exam. –Quiz on Friday –Enzyme mechanisms

Lecture 17

– Exams in Chemistry office with M’Lis. Please show your ID to her to pick up your exam.

– Quiz on Friday– Enzyme mechanisms

Terms to review for enzymes

• Cofactor

• Coenzyme

• Prosthetic group

• Holoenzyme

• Apoenzyme

• Lock and Key

• Transition analog model

• Induced fit

• Active site, binding site, recognition site, catalytic site

Catalytic Mechanisms

• Acid-base catalysis

• Covalent catalysis

• Metal ion catalysis

• Proximity and orientation effects (ex. anhydride)

• Preferential binding of the transition state complex

General Acid-Base Catalysis

• Large number of possible amino acids

• Requires that they can accept and donate a proton

• Glu, Asp

• Lys, His, Arg

• Cys, Ser, Thr

• Also can include metal cofactors (metal ion catalysis)

• Example can be observed in RNAse

Figure 15-2 The pH dependence of Vmax/KM in the RNase A–catalyzed hydrolysis of cytidine-2,3 -cyclic

phosphate.

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Example in book: RNAse (p. 499)

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•His12 acts as general base-takes proton from RNA 2’-OH-making a nucleophile which attacks the phosphate group.

•His119 acts as a general acid to promote bond scission.

•2’,3’ cyclic intermediate is hydrolyzed through the reverse of the first step-water replaces the leaving group. His12 is the acid, His119 acts as the base

RNAse mechanism

Covalent catalysis

• Rate acceleration through the transient formation of a catalyst-substrate covalent bond.

• Example-decarboxylation of acetoacetate by primary amines

• Amine nucleophilically attacks carbonyl group of acetoacetate to form a Schiff base (imine bound)

Figure 15-4 The decarboxylation of acetoacetate.

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uncatalyzed

Catalyzed by primary amine

Covalent catalysis

• Made up of three stages

1. The nucleophilic reaction between the catalyst and the substrate to form a covalent bond.

2. The withdrawal of electrons from the reaction center by the now electrophilic catalyst

3. The elimination of the catalyst (reverse of 1.)

• Nucleophilic catalysis - covalent bond formation is limiting.

• Electrophilic catalysis-withdrawal of electrons is rate limiting

Covalent catalysis

• Nucleophilicity is related to basicity. Instead of abstracting a proton, nucleophilically attacks to make covalent bond.

• Good covalent catalysts must have high nucleophilicity and ability to form a good leaving group.

• Polarized groups (highly mobile e-) are good covalent catalysts: imidazole, thiols.

• Lys, His, Cys, Asp, Ser

• Coenzymes: thiamine pyrophosphate, pyridoxal phosphate.

Covalent Catalysis• Form transient, metastable intermediates that can supply

bond energy into the reaction.

Serine

Side chainNH

RC-O-CH2-CH

O

COO-(acyl ester)

ChymotrypsinTrypsinElastaseacetylcholinesterase

structures Examples

Serine

-O-P-O-CH2-CH

O

(phosphoryl ester)

O

NH

COO-

PhosphoglucomutaseAlkaline phosphatase

Covalent Catalysis

Cysteine

GroupNH

RC-S-CH2-CH

O

COO-(acyl cysteine)

Papain3-PGAL-DH

structures Examples

Histidine

-O-P-N

O

(phosphoryl imidazole)

O

NHCOO-

Succinate thiokinase CH

Covalent Catalysis

Lysine

GroupNH

R-C=N-(CH2)4-CH

R'

COO-(Schiff base)

AldolaseTransaldolase

structures Examples

Metal ion catalysis

• Almost 1/3 of all enzymes use metal ions for catalytic activity. 2 main types:

1. Metalloenzymes-have tightly bound metal ions, mmost commonly transition metal ions such as Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, or Co3+

2. Metal-activated enzymes-loosely bind metal ions form solution-usually alkali or alkaline earth metals-Na+, K+, Ca2+

Metal ion catalysis

• Three ways for catalysis

1. Binding to substrates to orient them properly for the reaction

2. Mediating oxidation-reduction reactions through reversible changes in the metal ion’s oxidation state

3. Electrostatically stabilizing or shielding negative charges.

Serine Hydrolases (Proteases)

• Chymotrypsin, trypsin and elastase.

• All have a reactive Ser necessary for activity.

• Catalyze the hydrolysis of peptide (amide) bonds.

• Chymotrypsin can act as an esterase as well as a protease.

• Study of esterase activity provided insights into the catalytic mechanism.

NO2

p-Nitrophenylacetate

CH3 O

O

C

CH3 O-

O

C NO2-O

p-NitrophenolateAcetate

ChymotrypsinH2O

2H+

+

Serine Hydrolases (Proteases)

• Reaction takes place in 2 phases

1. The “burst phase”-fast generation of p-nitrophenolate in stoichiometric amounts with enzyme added

2. The “steady-state phase”-p-nitrophenolate generated at reduced but constant rate; independent of substrate concentration.

Figure 15-18 Time course of p-nitrophenylacetate hydrolysis as catalyzed by two different concentrations of chymotrypsin.

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NO2

p-Nitrophenylacetate

CH3 O

O

C

CH3 O-Enzyme

O

C

NO2-O

p-Nitrophenolate

Acyl-enzyme intermediate

Chymotrypsin

H2O2H+

+ Enzyme

CH3 O-

O

CAcetate

+ Enzyme

SLOW

FAST

Chymotrypsin

• Follows a ping pong bi bi mechanism.

• Rate limiting step for ester hydrolysis is the deacylation step.

• Rate limiting step for amide hydrolysis is first step (enzyme acylation).

Identification of catalytic residues

• Identified catalytically important residues by chemical labeling studies.

• Ser195-identified using diisopropylphospho-fluoridate (DIPF)

• Irreversible!

(active Ser)-CH2OH

F-P=O

O

Diisopropylphospho-fluoridate (DIPF)

O+

CH(CH3)2

CH(CH3)2

(active Ser)-CH2O -P=O

O

O

CH(CH3)2

CH(CH3)2

DIP-enzyme


• His57 was identified through affinity labeling

• Substrate analog with a reactive group that specifically binds to the active site of the enzyme forms a stable covalent bond with a nearby susceptible group.

• Reactive substrate analogs are sometimes called “Trojan horses” of biochemistry.

• Affinity labeled groups can be identified by peptide mapping.

• For chymotrypsin, they used an analog to Phe.

CH2ClCH3 C

O

NHS

OO

CH

CH2


Tosyl-L-phenylalanine chloromethyl ketone (TPCK)

Figure 15-19Reaction of TPCK with chymotrypsin to alkylate His 57.

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Homology among enzymes

• Bovine chymotrypsin, bovine trypsin and porcine elastase are highly homologous

• ~40% identical over ~240 residues.

• All enzymes have active Ser and catalytically essential His

• X-ray structures closely related.

• Asp102 buried in a solvent inaccessible pocket (third enzyme in the “catalytic triad”)

X-ray structures explain differences in substrate specificity

• Chymotrypsin - bulky aromatic side chains (Phe, Trp, Tyr) are preferred and fit into a hydrophobic binding pocket located near catalytic residues.

• Trypsin - Residue corresponding to chymotrypsin Ser189 is Asp (anionic). The cationic side chains of Arg and Lys can form ion pairs with this residue.

• Elastase - Hydrolyzes Ala, Gly and Val rich sequences. The specificity pocket is largely blocked by side chains of Val and a Thr residue that replace Gly residues that line the binding pocket of chymotrypsin and trypsin.

Figure 15-20a X-Ray structure of bovine trypsin.

(a) A drawing of the enzyme in complex.

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Figure 15-20b X-Ray structure of bovine trypsin. (b) A ribbon diagram of trypsin.

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Figure 15-20c X-Ray structure of bovine trypsin. (c) A drawing showing the surface of trypsin

(blue) superimposed on its polypeptide backbone (purple).

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Figure 15-21 The active site residues of chymotrypsin.

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Figure 15-22Relative positions of the active site residues insubtilisin, chymotrypsin, serine carboxypeptidase II, and

ClpP protease.

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Figure 15-23Catalytic

mechanism of the serine proteases.

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