Enzymes Lecture 4 Flashcards
This is a digestive enzyme lpsinocated in the small intestine
Chymotrypsin
Chymotrypsin catalyzes the hydrolytic of ______ bonds in proteins (a protease)
It is specific for peptide bonds adjacent to ______ AA residues
Peptide bonds
Aromatic (trp, phe, tyr)
What are the 3 steps of the chymotrypsin mechanism
General acid-base catalysis
• Covalent catalysis
• Transition-state stabilization
Does chymotrypsin catalyze a direct attack of water on the peptide bond?
What occurs?
No.
A transient covalent acyl enzyme intermediate is formed
The chymotrypsin reaction has two phases
Acylation and Deacylation
This step of the chymotrypsin mechanism cleaves the peptide bond and forms an ester linkage between peptide carbonyl carbon and the enzyme
Acylation
This phase of the chymotrypsin hydrolyzes the ester linkage and regenerates the nonacylated enzyme
Deacylation
What is the primary structure of chymotrypsin?
3 polypeptide chains linked by several disulfide bonds
Which three AAs are important for catalysis?
How do they occur in 3D structure?
His57, Asp102, Ser195
Grouped together
Evidence for a covalent acyl enzyme intermediate came from ______ kinetics
In this experiment, a small ester (p-nitrophenylacetate) was hydrolyzed by chymotrypsin.
Hydrolysis was determined by measuring the release of ____
The reaction proceeded with a rapid initial phase, which is which phase?
Then it was followed by a slower phase, which was which phase?
Pre-steady state kinetics
p-nitrophenol
Acylation
Deacylation
In terms of the effect of pH on chymotrypsin catalyzed reactions, a maximum rate is observed at about pH =
In fig 6-20a, the rate plotted is at low (subsaturing) [S] so it represents
Decreased Kcat at low pH results from protonation of _____
Where does the proton come from? (acid-base catalysis)
8
Kcat/Km
His57
Ser
A plot of 1/Km also shows pH dependence of chymotrypsin, there is a decline after pH =
The α-amino group of _____ loses a proton at a high pH
It is the __ terminal end of one of chymotrypsin’s chains
8
Ile16
Amino terminal
Ser, His, and asp are linked in a hydrogen bonding network called a
Catalytic triad
In acylation (at the beginning) the substrate (S) is a polypeptide containing an aromatic amino acid ADJACENT to the ___ bond to be cleaved
The polypeptide binds to the _______ of chymotrypsin
The aromatic amino acid residue next to the peptide bond to be cleaved sits in a ___ pockey on the enzyme
Peptide bond
Active site
Hydrophobic
What is the nuclephile in the acylation phase?
Ser195
When a peptide substrate binds to chymotrypsin, the hydrogen bond between ___ and ____ is compressed, the pKa of his increases from 7 to 12
Acting as a general base, ____ can remove the proton from the ser OH
Asp and His
His
In the acylation phase, interaction of ser and his creates a strong nucleophilic alkoxide ion on ____
The positively charged __ group of his is stabilized by the negatively charged ___ group of asp
Ser 195
Carboxylate
In the second step of the acylation phase, formation of a ____ ______ state occurs
The alkoxide ion from Ser 195 attacks the peptide ___ group forming this short lived acyl-enzyme intermediate
Tetrahedral transition state
Carbony
Note in the mechanism figure that a short-lived
negative charge on the carbonyl oxygen of the
substrate is generated. This charge is stabilized by H bonds from the amide group of gly 193 and one other residue.
!
Note - the H bond from Gly 193 is present ONLY in the _____ and in the _____ states for its formation and breakdown
What it do for these states?
Intermediate and transition
Reduces the energy required to reach them
Gly 193 reducing the energy required to reach intermediate and transition states is an example of ________ energy in catalysis
Binding energy
In step 3 of the acylation mechanism, what occurs?
Reformation of a ____ bond between the carbonyl oxgen and carbon displaces the bond between the carbon and amino group of the peptide linkage, breaking the ______
The amino group of the peptide bond is protonated by _____ and diffuses away from the enzyme’s active site, leaving an acyl-enzyme intermediate with it’s aromatic AA residue in the hydrophobic pocket of the enzyme
Formation of the acyl -enzyme intermediate
His 57
In step 4 (deacylation phase), an incoming water molecule is deprotonated by general ___ catalysis
The imidazole group of _____ accepts a proton from
This results in a ___ nucleophile
Base
His57, Water
Hydroxide
In step 5 (deacylation phase), what occurs?
The ___ ion attacks the ___ linkage of the acyl enzyme
This generates a short lived tetrahedral intermediate
What once again becomes negatively charged?
Formation of a second tetrahedral transition state
Hydroxide attacks the ester linkage
The oxygen
In step 6, the tetrahedral intermediate formed in step 5 collapses and forms a ____ ______
What is displaced?
Carboxylate anion
Ser 195
In step 7, diffusion of the second product from the active site of the enzyme regenerates the free enzyme.
!
What does the triad of AAs and the H-bonding groups do for the chymotrypsin reaction?
How?
Lowers the activation energy
Stabilizing transition states
What group of cysteine in thiol proteases like papain functions in a similar way to the hydroxyl group of serine?
The binding of a metal ion to the imidazole of his 57 or the thiolate of cys in papain will do what?
Thiol (SH) group
Inhibit the reaction
These are common non-competitive inhibitors of many enzymes
These metal ions form strong complexes with
neutral and anionic groups of amino acid side
chains which are important to enzyme
catalysis (such as the imidazole of his or the
thiolate of cys)
Heavy metal ions (Pb2+, Ag+, Hg+)
________ ,a bisubstrate enzyme, catalyzes the reversible reaction of β-D-glucose to glucose-6-phosphate with Mg·ATP converted to Mg·ADP
Yeast hexokinase
ATP and ADP always bind to enzymes as a complex with the metal ion ______
The γ-phosphoryl of ATP is transferred to the
OH at C-6 of glucose.
Water freely enters the enzyme active site,
but hexokinase favors reacting with the OH of
glucose over the OH of water by a factor of
106.
Mg2+
!
Hexokinase disciminates between water and glucose because of a ______ in the enzyme when the correct substrate binds
This is a good example of _____
When glucose is absent, hexokinase is in an inactive conformation
Conformational Change
Induced fit
When glucose and Mg·ATP bind, the binding
energy induces a conformational change to the catalytically active form of _____
hexokinase
This is a glycolytic enzyme
It catalyzes the reversible _____ of 2-phosphoglycerate to phosphoenolpyruvate
What type of catalysis is this?
How many steps?
Then enolic intermediate is stabilized by what?
Enolase
Dehydration
Metal ion catalysis
Two steps
Two Mg2+ ions
A natural bacterial agent found in tears and egg whites
Lysozyme
This enzyme cleaves the B1–>4 glycosidic C-O bond between two different sugar residues the the molecule N acetylmuramic acid and N acetylglucosamine.
What type of reacion is it?
Which type is supported?
Lysozyme
Nucleophilic substitution
SN2
