Lesson 7: Enzyme Mechanism Flashcards by Hannah Añonuevo

Types of Catalytic Strategies

Covalent Catalysis
Nucleophilic Catalysis
Electrophilic Catalysis
Specific Acid-Base
General Acid-Base
Metal Ion Catalysis

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Covalent Catalysis

-Forms a transient covalent bond.
-Can cleave bonds between the substrate.
-Retrieve catalyst via hydrolysis

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Nucleophilic Catalysis

-Nucleophilic centers (Nu-) on the enzyme attack electrophilic targets on substrate

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Electrophilic Catalysis

-Involve coenzymes that form electrophiles (El+)

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Specific acid-base

-Involves H+ or OH-
-Not dependent on buffer concentration

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General acid-base

-H+ or OH- is formed in the transition state by another molecule

-Use acidic polar amino acids

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General Acid Catalysis Mechanism

Carbonyl bond attacks the H from the enzyme
To maintain two bonds in O of carbonyl, one bond transfers to the C-H bond
CH3 becomes C=CH2 as other H gets removed.
Acid reacts with water to retrieve the enzyme.
Product+Enzyme+OH-

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General Basic Catalysis Mechanism

Base gets the H from CH3
H bond connecting to CH2 moves to the latter, forming C=CH2
One bond from the carbonyl group gets transferred to oxygen, becoming O-.
O- is stabilized by H+.
Product+Base+H+

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Metal ion catalysis

-Use of metals as redox cofactors
-Stabilize formation of negative (-) charge
-Act as a nucleophile at neutral pH

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Metalloenzymes

Metal ion tightly bound

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Metal-activated enzymes

Metal ion loosely bound

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Lysozyme

-Cleaves peptidoglycan layer of the cell wall of bacteria

-Hydrolyzes glycosidic bond between N-acetylmuramic acid (NAM or MurNAc) and N-acetylglucosamine (NAG or GlcNAc)

-Mechanism involves two successive SN2 reactions

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Gram-negative peptidoglycan layer

Has an outer wall covering the thin peptidoglycan layer

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Gram Positive Peptidoglycan Layer

No outer layer, peptidoglycan layer is exposed and easier to access when staining

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Binding Site

-Accomodate 6 sugar residues
-Cleavage occurs between D and E sites
-Sugar (NAM) is distorted at D site
-Alternate between NAM and NAG

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Catalytic Residues for Lysozyme

-Asp52(D52): Nucleophile (undergoes Nu covalent catalysis)

-Glu35(E35): General Acid-Base

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Chymotrypsin

Cuts C-side of Phe, Trp, Tyr, and Leu

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Proteases

Proteins that hydrolyze peptide bonds

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Serine Protease

Use Ser as the nucleophile

Oxyanion hole

-Very reactive
-Tetrahedal intermediate

Nucleophile

Ser195

Catalytic Triad

Ser195- Nucleophile
Asp102- Orients H57
His57- General Acid-Base Catalyst

S1 pocket (hydrophobic pocket)

-For specificity
Cuts nuetral nonpolar amino acids

Burst Kinetic Study of Chymotrypsin

Acylation
-Faster because there’s a lot of enzymes that can cleave the bond
Deacylation
-Slower because it waits for the presence of water to go back to its original form.

Chymotrypsin Mechanism

1. Nucleophilic attack of Ser195 to peptide bond, undergoes covalent catalysis; forms a tetrahedal intermediate. 2. Acylation, amine group separated from the carbonyl with catalyst attached to it. Amine group is stabilized by H from His57. 3. Deacylation, OH- of water (other H comes from His57) acts as a nucleophile to form tetrahedal intermediate, breaking its bond with the enzyme. 4. O- in the enzyme is stabilized using His57. 5. Product formation and retrieval of enzyme success!

Substrate level control

Rate depends on substrate availability -No suubstrate, no enzyme activity

Genetic Control

Determines the amount of gene present -Gene is responsible for producing enzymes.

Induced Enzymes

Enzymes produced by the gene to accomodate for the number of substrate (can be more or less enzymes)

Feedback control/allosteric control

-Regulation at committed steps (irreversible reaction) of pathways -Inhibition by one of its pathway's product -Mediated by allosteric enzymes wherein it binds in the alloseteric site since they are not complementary

Covalent modification

Reversible covalent attachment of a chemical group

Zymogems (proenzymes)

-Inactive precursors of enzymes -Converted into their active forms via proteolytic enzymes -Activation via irreversible modification

Isoenzymes

Enzymes with slightly different subunits

Allosteric Regulation Properties

-Sigmoidal Michealis-Menten Curve -Cooperativity, has more than one subunit

T-State

-Tense/taut -low affinity state (substrate)

R-state

-Relaxed -High affinity state (substrate)

Covalent Modification Various Types of Reactions

-Phosporylation -Uridylation -Methylation -Adenylation -ADP-ribosylation -Disulfide bond formation

Kinases

-Adds P group to OH-containing amino acids (such as Ser, Thr, Tyr) -Transferase -Phosporylated enzyme is not equal to active enzyme

Phosphatases

-Removes P group -Hydrolase (use water to cleave P group and enzyme) -Dephosporylated enzyme is not equal to active enzyme

Enteropeptidase

-Controls trypsin production

Trypsin

-Cuts C-side of Arg and Lys (Basic) -S1 Speicificity: Polar acidic (opposite charges) -Activates trypisinogen to create more trypsin and other enzymes

Proelatase active enzyme

Elatase

Prolipase active enzyme

Lipase

Chymotrypsinogen active enzyme

Chymotrypsin

Procarboxypeptidase

Carboxypeptidase