biochem chapter 8 exam 2

Affinity Labeling of Histidine 57

inactivates chymotrypsin

decreasing the overall number of active enzyme molecules in contrast with competitive inhibition, cannot be overcome by increasing the substrate concentration.

noncompetitive inhibition

substrate is not prevented from binding to the enzyme

noncompetitive inhibition

uncompetitive inhibitor removes

some ES from the equilibrium

There are three common types of reversible inhibition:

-competitive inhibition -uncompetitive inhibition -noncompetitive inhibition

Irreversible inhibitors can be classified into 4 categories:

-group-specific reagents -affinity labels (substrate analogs) -suicide inhibitors -transition-state analogs

-covalently modify active-site residues -are structurally similar to an enzyme's substrate (they more specific for an enzyme's active site than are group-specific reagents)

Affinity labels or substrate analogs

Many reactions include 2 distinct substrates. In such cases, the reaction rate may be considerably enhanced by bringing the two substrates into proximity and in the proper orientation on a single binding surface of an enzyme.

Catalysis by Approximation and Orientation

Chymotrypsin contains 3 important aminoacids in it's active site (Catalytic Triad)

Asp His Ser

acts by covalently modifying the enzyme cyclooxygenase (the same enzyme inhibited by ibuprofen), reducing the synthesis of inflammatory signals.

Aspirin

The unstable tetrahedral-intermediate bearing a negative charge on the O atom fits within a pocket on the enzyme called the oxyanion hole, and it is stabilized by hydrogen bonds contributed by the amide groups of 2 peptide bonds in the chymotrypsin backbone. One of these hydrogen bonds (contributed by Gly193) is present only in this intermediate and in the transition states for its formation and breakdown; it reduces the energy required to reach these states. This is an example of the use of binding energy in catalysis.

Chymotrypsin Mechanism: the oxyanion hole

The specificity pocket of chymotrypsin

Examination of the three-dimensional structure of chymotrypsin with substrate analogs and enzyme inhibitors revealed the presence of a deep, relatively hydrophobic pocket, called the S1 pocket, into which the long, uncharged side chains of phe and trp can fit. The binding of an appropriate side chain into this pocket positions the adjacent peptide bond into the active site for cleavage Notice that many hydrophobic groups line the deep specificity pocket (shown in green). Also notice that the active-site serine residue (ser 195) is positioned to cleave the peptide backbone between the residue bound in the pocket and the next residue in the sequence. The key amino acids that constitute the binding site are identified.

acts by covalently modifying the enzyme transpeptidase, thereby preventing the synthesis of bacterial cell walls and thus killing the bacteria (p. 132).

Penicillin

Some irreversible inhibitors are important drugs:

Penicillin and Aspirin

is an important process in living systems. Proteins ingested in the diet must be broken down into small peptides and amino acids for absorption in the intestine. After a protein is no longer needed in the cell, it must be degraded so that their constituent amino acids can be recycled for the synthesis of new proteins.

Protein turnover

chymotrypsin cleaves peptide bonds selectively on the

carboxyl-terminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine (shaded orange).The red bonds indicate where chymotrypsin most likely acts

Substrate binds to

an enzyme's active site to form an enzyme-substrate complex.

the activity-versus-pH curves

are due to several ionizable groups.

The activity of most enzymes displays a

bell-shaped curve when examined as a function of pH.

A noncompetitive inhibitor

binds at a different site and does not prevent the substrate from binding.

A competitive inhibitor

binds at the active site and thus prevents the substrate from binding.

TPCK binding

binds at the chymotrypsin active site and modifies an essential His residue

An uncompetitive inhibitor

binds only to the enzyme-substrate complex.

Enzyme kinetics can sometimes be monitored with

chromogenic (colored) substrates or products.

Proteolytic enzyme example

chymotrypsin

The mechanism observed with the chromogenic substrate mimics what happens with normal protein substrates. The two steps are explained by the formation of a covalently bound enzyme-substrate intermediate

chymotrypsin mechanism

The enzymes of the pancreatic secretion of the upper small intestine, such as

chymotrypsin, have pH optima near pH 8, in keeping with the pH of the intestine

In ________the I competes with the S for the active site. The hallmark of ________ is that it can be overcome by a sufficiently high concentration of substrate (Vmax is unchanged) The effect of a ________inhibitor is to increase the apparent value of KM, called kmapp

competitive

substrate is prevented from binding to the enzyme

competitive and uncompetitive inhibition

At any given inhibitor concentration, _________________ can be relieved by increasing the substrate concentration. Under these conditions, the substrate "outcompetes" the inhibitor for the active site

competitive inhibition

the inhibitor resembles the substrate and binds to the active site of the enzyme ⇒ the substrate is prevented from binding to the same active site An enzyme can bind substrate (forming an ES complex) or inhibitor (EI), but not both (ESI)

competitive inhibition

diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate

competitive inhibitor

For enzymes that exhibit Michaelis-Menten kinetics measurements of the rates of catalysis at different concentrations of substrate and inhibitor serve to distinguish the 3 types of reversible inhibition:

competitive: same vmax, increased Km uncompetitive:decrease Vmax, decreased Km noncompetitive: decreased Vmax, Same Km

the active site contains a reactive group (usually a powerful nucleophile) that becomes temporarily covalently modified in the course of catalysis. Example: chymotrypsin

covalent catalysis

an antibiotic, functions at low concentrations as a noncompetitive inhibitor of a bacterial proteolytic enzyme (collagenase). Inhibition of this enzyme prevents the growth and reproduction of bacteria that cause gum (periodontal) disease.

doxycycline

The rate of enzymatic catalysis is affected by

environmental parameters

a molecule other than water plays the role of a proton donor or acceptor. Chymotrypsin uses a histidine residue as a base catalyst to enhance the nucleophilic power of serine

general acid-base catalysis

a substrate can bind to the enzyme-inhibitor complex as well as to the enzyme alone. In either case, the enzyme-inhibitor-substrate complex does not proceed to form product. The value of Vmax is decreased to the new value (the inhibitor simply lowers the concentration of functional enzyme), whereas the value of KM is unchanged.

noncompeitive (mixed) inhibition

the inhibitor and substrate can bind simultaneously to an enzyme molecule at different binding sites ↓ Vmax; unchanged KM

noncompetitive inhibition

can be obtained by examining product formation within milliseconds of mixing the enzyme and substrate.

pre-steady state kinetics data

catalyzed by a large class of enzymes called proteolytic enzymes or proteases. These enzymes cleave proteins by a hydrolysis reaction—the addition of a molecule of water to a peptide bond.

protein breakdown

The characteristics of competitive inhibition, are

same Vmax; ↑ KM (the enzyme requires more substrate to achieve ½ Vmax)

Chymotrypsin uses ______________to attack the unreactive carbonyl group of the protein substrate to be hydrolyzed, and becomes covalently attached to the substrate briefly in the course of catalysis.

ser 195 (a powerful nucleophile)

Hydrolysis of the Acyl-Enzyme intermediate with release of product

slow phase stage 2

A metal ion may:

stabilize a negative charge on a reaction intermediate. increase the acidity of a nearby molecule, such as water. bind to the substrate, increasing the number of interactions with the enzyme and thus the binding energy.

Under_________________, the cleavage of the substrate obeys Michaelis-Menten kinetics.

steady state conditions

The tetrahedral intermediate (formed in 2) collapses with the breaking of the peptide bond to generate the acyl-E (3). This step is facilitated by the transfer of the proton from the positively charged histidine residue, now acting as a general acid catalyst, to the amino group of the substrate formed by cleavage of the peptide bond. The amine component is now free to depart from the E (4), completing the first stage of the hydrolytic reaction: acylation of the E at Ser 195.

step 3 and 4 of chymotrypsin mechanism

The next stage—deacylation of the enzyme—begins when a water molecule takes the place occupied earlier by the amine component of the substrate (5). The ester group of the acyl-enzyme is now hydrolyzed by a process that essentially repeats steps 2 through 4. Again acting as a general base catalyst, His 57 draws a proton away from the water molecule. The resulting OH− ion attacks the carbonyl carbon atom of the acyl group, forming a tetrahedral intermediate (6).

step 5 and 6 of chymotrypsin mechanism

The tetrahedral intermediate (6) breaks down to form the carboxylic acid product (7). Finally, the release of the carboxylic acid product (8) readies the enzyme for another round of catalysis.

step 7 and 8 of chymotrypsin mechanism

chemically modified substrates These molecules provide researchers with the most-specific means of modifying an enzyme's active site. The inhibitor binds to the enzyme just like a substrate, and is initially processed by the normal catalytic mechanism The mechanism of catalysis then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification. Penicillin, the first antibiotic discovered, is a suicide inhibitor of the enzyme that synthesizes bacterial cell walls: Glycopeptide transpeptidase

suicide inhibitors

Enzyme activity can be modulated by

temperature, pH, and inhibitory molecules

for most enzymes, there is a temperature optimum

the catalytic activity decreases on both sides of the temperature optimum

The uncompetitive inhibitor's binding site is created only when

the enzyme binds the substrate.

the pH at which enzymes display maximal activity—varies with the enzyme and is correlated with the usual environment of the enzyme.

the optimal pH

The enzyme tyrosinase is part of the pathway that synthesizes

the pigment that results in dark fur

The interactions between the 3 amino acids in the catalytic triad bring about the active site:

the side chain of Ser195 (—OH group ) is hydrogen bonded to the N of the imidazole ring of His 57 the —NH group of the His 57 imidazole ring is, in turn, hydrogen bonded to the carboxylate group of Asp 102 (—COO—)

the enzymatic activity decrease above

the temperature optimum is abrupt: when the temperature increases beyond a certain point, the weak bonds maintaining the three-dimensional structure are not strong enough to withstand the polypeptide chain's thermal jostling and the protein loses the structure required for activity ⇒ the protein is denatured

Formation of the Acyl-Enzyme intermediate

(fast phase) stage 1

If the pH is raised

(less H+, more OH+), the —NH3+ group loses a H+ to OH+, becoming a neutral —NH2 group, and the enzyme activity is diminished.

If the pH is lowered

(the H+ concentration increases), the −COO− groups will gradually be converted into −COOH groups with a loss of enzyme activity.

are potent inhibitors of enzymes

transition state analogs

is distinguished by the fact that the inhibitor binds only to the enzyme-substrate complex

uncompetitive inhibition

the inhibitor binds only to the ES complex

uncompetitive inhibition

cannot be overcome by the addition of more substrate.

uncompetitive inhibitor

The characteristics of uncompetitive inhibition, are:

↓ Vmax: 𝑽_𝒎𝒂𝒙^𝒂𝒑𝒑 (reduced Vmax is called Vmax apparent) ↓ KM: 𝑲_𝑴^𝒂𝒑𝒑(reduced KM is called KM apparent

A group-specific reagent is diisopropylphosphofluoridate (DIPF) DIPF modifies a ser residue in the enzyme active site

⇒ inhibits the proteolytic enzymes such as chymotrypsin DIPF can inhibit an enzyme by covalently modifying a crucial serine residue

-react with the specific R groups of amino acids: -covalently modify active-site residues -are NOT structurally similar to an enzyme's substrate

Group-specific reagents (DIFP)

Enzyme inhibition can be either reversible or irreversible

In contrast with irreversible inhibition, reversible inhibition is characterized by rapid dissociation of the enzyme-inhibitor complex

Tyrosinase has a low tolerance for heat in Siamese cats

It is inactive at normal body temperatures but functional at slightly lower temperatures. The extremities of a Siamese cat are cool enough for tyrosinase to gain function and produce pigment.

Reversible Inhibitors Are

Kinetically Distinguishable

Metal ions are required cofactors for many of the enzymes.

Metal ion catalysis

Aspartic Acid is Part of a Catalytic Triad That includes

Serine and Histidine

Chymotrypsin Mechanism the acyl group of the substrate becomes covalently attached to ser 195 of the enzyme: acyl-enzyme intermediate

Step 1(FAST STEP):

Chymotrypsin Mechanism the acyl-enzyme intermediate is hydrolyzed to release the carboxylic acid component of the substrate and regenerate the free enzyme):

Step 2 (SLOWER STEP):

an affinity label for chymotrypsin. ______ binds at the active site and then reacts irreversibly with a histidine residue at that site, inhibiting the enzyme.

TPCK

Affinity labeling: react chymotrypsin with a molecule that resembles a substrate binds to the active site forms a stable covalent bond with a group on the enzyme that is in proximity.

TPCK meets these criteria: The phenylalanine side chain of TPCK enables it to bind specifically to chymotrypsin. The reactive group in TPCK is the chloromethyl ketone, which covalently modifies one of the ring nitrogens of histidine 57 The TPCK derivative of chymotrypsin is enzymatically inactive.

How does this arrangement of residues lead to the high reactivity of serine 195?

The His residue serves to (1) position the Ser side chain and to (2) polarize its hydroxyl group so that it is ready for deprotonation: In the presence of the substrate, His-57 accepts the proton from the Ser-195 hydroxyl group ⇒ His acts as a general base catalyst. The withdrawal of the proton from the hydroxyl group generates an alkoxide ion (O-) on the Ser, which is a much more powerful nucleophile than a hydroxyl group. The Asp residue helps orient the His residue and make it a better proton acceptor through hydrogen bonding and electrostatic

Irreversible inhibitors that covalently bind to an enzyme provide a way for elucidating the mechanism of enzymes.

The first step in determining the chemical mechanism of an enzyme is to determine which functional groups are required for enzyme activity. Irreversible inhibitors modify functional groups, which can then be identified.

an uncompetitive inhibitor of an enzyme in the biosynthetic pathway for aromatic amino acids in plants. The plant dies because it lacks these amino acids.

The herbicide glyphosate, also known as Roundup

functions in the highly acidic environment of the stomach, where the pH is between 1 and 2

The protein-digesting enzyme pepsin

Chymotrypsin mechanism After substrate binding (1), the reaction begins with the O atom of the side chain of Ser 195 making a nucleophilic attack on the carbonyl C atom of the target peptide bond (2) ⇒ E-S intermediate.

There are 4 atoms bonded to the carbonyl C atom: very short-lived unstable tetrahedral-intermediate bears a negative charge on the O atom of the carbonyl group of the substrate

In uncompetitive inhibition,

This enzyme-substrate-inhibitor complex, ESI, does not proceed to form any product The inhibitor removes some ES from the equilibrium, the concentration of active ES is smaller than in the absence of I ⇒ -Vmax is reduced (↓ Vmax: 𝑽_𝒎𝒂𝒙^𝒂𝒑𝒑) -less S is required to form half of the maximal concentration of ES, resulting in a reduction of the value of KM (↓ KM: 𝑲_𝑴^𝒂𝒑𝒑)

N-acetyl-L-phenylalanine p-nitrophenyl ester is a chromogenic substrate analog

When used as a substrate, the enzyme generates a yellow product (p-nitrophenolate), detected by using a spectrophotometer

Imagine an enzyme that requires the ionization of both

a glutamic acid residue (—COO− ) and a lysine residue (—NH3+) at the active site for the enzyme to be functional.

Some competitive inhibitors commonly used as drugs include

ibuprofen, which inhibits a cyclooxygenase that helps to generate the inflammatory response, and statins, which inhibit the key enzyme in cholesterol synthesis.

Treatment with group-specific reagents that modify serine residues, such as DIPF, was found to

inactivate the enzyme irreversibly Despite the fact that the enzyme possesses 28 ser residues, only Ser 195 was modified, resulting in a total loss of enzyme activity

temperature

increases the rate of most reactions ↑ Brownian motion of the molecules which ↑ interactions between an enzyme and substrate and ↑ the number of molecules attaining the transition state).

The binding of specific small molecules and ions can

inhibit the activity of many enzymes. In addition to the evolutionarily derived signal molecules, many drugs and toxic agents act by inhibiting enzymes.

interact specifically with the 3-D structure of the enzyme also can affect enzyme activity.

inhibitors

The reaction between chymotrypsin and N-acetyl-L-phenylalanine p-nitrophenyl ester produced an

initial rapid burst of colored product (pre-steady state), followed by a slower phase as the (steady state). These results suggest that hydrolysis proceeds in 2 steps. The burst is observed because the first step is more rapid than the second step

dissociates very slowly from its target enzyme because it has become tightly bound to the enzyme, either covalently or noncovalently.

irreversible inhibitor