Lesson 10: Enzyme Reaction Mechanisms

enzymes

biological catalysts

ribozymes

enzymes made of RNA

active site

3D cleft/crevice containing catalytic residues; substrate binding + reaction occurs here

catalytic residues

residues in active site that facilitate reaction

specificity conferred by (2)

geometric and electronic complimentarity

geometric complimentarity

substrate has to physically fit properly

electronic complimentarity

proper charges/partial charges in specific spots

lock and key model

enzyme and substrate fit together like a lock and key

induced fit model

active site has some complimentarity, but upon substrate binding undergoes conformational change to fit substrate better

which model is currently accepted (lock and key or induced fit)

induced fit

cofactors

additional molecules required for some enzymes to function

types of cofactors

metal ions, coenzymes

types of coenzymes

prosthetic group (permanently bound), transient (binds, facilitates rxn, leaves)

apoenzyme

enzyme missing its required cofactor --> inactive

holoenzyme

enzyme with its cofactor --> active

enzymes are classified by...

the type of reaction they catalyze

oxidoreductase

transfer of H or O (oxidation/reduction)

transferase

transfer of functional groups

hydrolase

formation of 2 products by hydrolyzing a substrate

lyase

cleavage without using H2O

isomerase

transfer of groups within a molecule (intramolecular rearrangement)

ligase

formation of bonds using ATP cleavage

enzymes only catalyze __ reactions

spontaneous, exergonic

transition state

activated form of a molecule that has undergone partial chemical reaction; highest energy point on a reaction coordinate (energy barrier)

activation energy (symbol and definition)

ΔG‡; energy required to reach transition state

enzymes catalyze by __ __ energy, this increases __

lowering activation; rate

enzymes facilitate (what state)

the transition state

enzymes DON'T affect (2)

ΔG, Keq

enzyme catalytic strategies (3)

acid-base catalysis, covalent catalysis, metal ion catalysis

acid-base catalysis

one or more residues in the active site act as proton donors/acceptora

most common residue in acid-base catalysis

His

RNAase mechanism

draw it!!

covalent catalysis (2 steps)

acylation --> deacylation

acylation

first step of covalent catalysis; nucleophile attacks substrate --> releases part of substrate, forms a covalent bond with other part of substrate

acyl-enzyme intermediate

covalently bound enzyme to substrate

deacylation

second step of covalent catalysis; water cleaves bond between enzyme and substrate

nucleophilic catalysis

another name for covalent catalysis

characteristics of covalent catalysis catalytic residues

usually nucleophilic side chain; negative charge or unshared e-

characteristics of attack site on substrate (in covalent catalysis)

electrophilic; has carbonyl group or + charge (usually amide or ester)

metal ion catalysis

uses metal ion cofactors

metal ions can... (3)

1) deprotonate substrate --> reactive nucleophile 2) act as electrophiles --> stabilize charge on intermediate 3) hold substrate in favorable position

why are metal ions better than protons (metal ion catalysis)

act the same, but concentration is not affected by pH

chymotrypsin is (type of enzyme)

serine protease

serine protease

family of enzymes that cleave peptide bonds using both acid-base and covalent catalysis

serine protease structural motifs (3)

1) binding pocket 2) catalytic triad 3) oxyanion hole

binding pocket

confers peptide bond specificity; shape / charge specific for certain side chains

catalytic triad

Ser, His, and Asp arranged close in 3° structure

oxyanion hole

nucleophilic Ser and Gly sit above active site to stabilize oxyanion intermediate

catalytic triad mechanism (chymotrypsin)

Asp's - charge makes His side chain more electrophilic --> His strips H+ from Ser --> negative charge on Ser acts as a nucleophile

chymotrypsin mechanism

draw it!!

enolase (overall reaction, when does this occur?)

converts 2PGA <--> phosphoenolpyruvate in glycolysis

enolase (catalysis type)

uses metal ion catalysis and acid-base catalysis

enolase cofactors and their function

2 Mg2+ cofactors stabilize 2 O- on 2-PGA

enolase catalytic residues (2)

Lys, protonated Glu (neutral)

enolase steps

Lys acts as a base, taking H+ from 2-PGA, forming a C-C double bond; carbanion stabilized by Mg2+ --> carbanion resolves, moving C-C double bond --> Glu acts as acid, donating H+ to 2-PGA's OH group --> OH leaves as H2O, forming phosphoenolpyruvate product

enolase mechanism

draw it!!

which residue acts as a base in enolase? what does it do?

Lys; takes H+ from 2-PGA to form C-C double bond

which residue acts as an acid in enolase? what does it do?

Glu; donates H+ to 2-PGA's OH group, OH leaves as H2O