Biochem Chapter 9: Catalytic Strategies Full

Metal ion catalysis info

1. A metal ion may serve as an electrophilic catalyst, stabilizing a negative charge on a reaction intermediate 2. The metal ion may generate a nucleophile by increasing the acidity of a nearby molecule (Ex. Water in the hydration of CO2 by carbonic anhydrase) 3. The metal ion may bind to the substrate, increasing the number of interactions with the enzyme and thus the binding energy (NMP Kinases)

hydrolysis by chymotrypsin takes place in two stages

1. Acylation to form the acyl-enzyme intermediate 2. Deacylation to regenerate the free enzyme

Lowering the activation energy

1. Covalent Catalysis 2. General acid-base catalysis 3. Metal ion catalysis 4. Catalysis by approximation

Restriction enzymes must show tremendous specificity by

1. Only cleaving DNA molecules that contain recognition sites without cleaving DNA molecule that lack these sites 2. Must not degrade the host DNA

The catalytic strategies used by four classes of enzymes

1. Serine Proteases 2. Carbonic Anhydrases 3. Restriction Endonucleases 4. Myosins, NMP Kinases

Catalysis entails zinc activation of a water molecule: mechanism for the hydration of carbon dioxide

1. Zn2+ generates OH− by facilitating the release of a H+ from water 2. CO2 binds to the active site and is positioned to react with OH− 3. The OH− attacks the CO2, generating HCO3− 4. The active site is regenerated with the release of HCO3− and binding of H2O

Which of the following is not the roles for Mg2 in myosin's hydrolysis of ATP? A) Binds to the enzyme and activates a water molecule B) Neutralizes partially the negative change on the triphosphate group of ATP C) Forms a stable conformation of ATP by binding to its phosphoanhydride "tail D) Provides potential binding points on the ATP for the enzyme to recognize

A

P-loop

A component of the nucleotide binding domain, the domain consists of a central beta sheet surrounded by alpha helices on both sides

substrate specificity: S1 pocket

A deep, relatively hydrophobic pocket, into which long uncharged side chains of residues can fit

General acid-base catalysis

A molecule other than water donates or accepts a proton

Acid base catalysis

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

Which of the following is NOT a way in which enzymes stabilize a transition state? A) Causing the temperature of the environment to increase B) Covalent catalysis C) Using bonding energy D) General acid-base catalysis E) Catalysis by approximation

A, Causing the temperature of the environment to increase

The P-loop of adenylate kinase interacts with the phosphoryl groups of

ATP

NMP Kinase: catalysis by approximation

ATP binding induces large conformational changes The enzyme holds its two substrates close together and appropriately oriented to stabilize the transition state that leads to the transfer of a phosphoryl group from the ATP to the NMP

Protease inhibitors

Are similar in structure to the peptide substrate of the enzyme that each inhibits Several important drugs are protease inhibitors

catalytic triad of chymotrypsin

Asp102, His57, Ser195

What do trypsin, chymotrypsin, and elastase all have in common? A) All contain Asn in the active site B) All contain a catalytic triad at the active site C) All need metal ion for the catalysis D) All bind hydrophobic amino acid E) All contain a hydrophilic substrate-binding pocket

B

The _______ _______ between enzyme and substrate is the free energy released in the formation of a large number of weak interactions between the enzyme and substrate

Binding Energy

Noncompetitive inhibitor

Binds either enzyme or enzyme-substrate complex Km is unchanged in the presence of this type of inhibitor, Vmax decreases

S1 pocket of chymotrypsin

Broad, nonpolar Binds to Phenylalanine, Tryptophan, Tyrosine

Which of the following is the most accurate statement regarding the enzyme chymotrypsin? A) chymotrypsin catalyzes the hydrolysis of peptide bonds on the carboxyl-terminal side of large positively charged amino acid B) Chymotrypsin is a member of a class of enzymes called phosphatases C) The catalytic mechanism of chymotrypsin involves a catalytic triad composed of an aspartate, a histidine, and a serine residue D) The catalytic mechanism of chymotrypsin involves the formation of a covalent thioester intermediate E) The catalytic mechanism of chymotrypsin involved a heme cofactor that interacts with a histidine residue in the active site

C, The catalytic mechanism of chymotrypsin involves a catalytic triad composed of an aspartate, a histidine, and a serine residue

NMP kinases bring two nucleotides, for example AMP and ATP, together to facilitate the transfer of a phosphoryl group from one nucleotide to the other (creating two ADP). The enzyme positions the adenosine triphosphate (ATP) such that the gamma phosphate group is placed adjacently to the phosphate group of the NMP kinase. This facilitates the transfer of the phosphate between the two. What example of catalysis is this?

Catalysis by Approximation

Carbonic anhydrases

Catalyzes the hydration and dehydration of CO2 Reaction is spontaneous at equilibrium

Elastase Specificity

Cleaves at the peptide bond after amino acids with small side chains (alanine and serine

Typsin Specificity

Cleaves at the peptide bond after residues with a long, positively charged side chains (arginine and lysine)

Proteases

Cleaves proteins by a hydrolysis reaction (the addition of a water molecule to a peptide bond)

specificity of chymotrypsin

Cleaves proteins on the carboxyl side of aromatic or large hydrophobic amino acids

Serine proteases

Common catalytic mechanism is characterized by the possession of a highly reactive serine residue that is essential for this enzyme activity, uses covalent modification as a catalytic strategy Cleaves peptide bonds selectively on the carboxyl-terminal side of the large hydrophobic amino acids (tryptophan, tyrosine, phenylalanine, and methionine)

The complete catalytic apparatus is assembled only within complex of _____ ______ molecules, ensuring specificity

Congate DNA

S1 pocket of trypsin

Contains Aspartate, negative charge Binds to Lysine and Arginine

S1 pocket of elastase

Contains Valine 216 and Valine 190, narrow nonpolar Binds to Glycine, Alanine, Valine

Elastase specificity pocket

Contains Valine and Threonine, binds to smaller and uncharged side chains

Trypsin specificity pocket

Contains aspartate, binds to Lysine or Arginine (positive amino acids)

In aspartate ammonia lyase, a deprotonated serine sidechain performs a nucleophilic attack on a carbonyl carbon of the substrate. What example of catalysis is this?

Covalent Catalysis

What type of reaction is catalyzed by proteases A) Oxidation-reduction B) Ligation C) Isomerization D) Hydrolysis E) Group transfer

D

Which group binds covalently to the substrate? A) Histidine 57 B) The specificity pocket C) The oxyanion hole D) Serine 195 E) Aspartic acid 102

D

All restriction enzymes catalyze the hydrolysis of

DNA phosphodiester bonds, leaving a phosphoryl group attached to the 5' end

____________ is crucial for catalysis because it locates the phosphoanhydride linkage near the magnesium, which is crucial for hydrolysis

Distortion

How is specificity determined by chymotrypsin? A) Interactions of the active site amino acids with the substrate B) Binding of the N-terminus amino acid at the active site C) Covalent binding of a His residue to the substrate D) Large conformational change of P-loop upon binding of substrate E) binding of the proper amino acid into a deep pocket on the enzyme

E

What metal ion is required by carbonic anhydrase for activity? A) Mn+2 B) Fe+2 C) Mg+2 D) Cu+2 E) Zn+2

E

Which of the following best describes the type of reaction catalyzed by a kinase A) Adenylylation B) Carboxylation C) Hydrolysis D) Phosphorolysis E) Phosphorylation

E

Myosins

Elongated structures with globular domains that harbor the active site, enzymes that use the energy of ATP hydrolysis to power the movement of molecules within the cell

How does the binding of the magnesium ion to the nucleoside affect catalysis?

Enhances the specificity of the enzyme-substrate interactions by enhancing binding energy

Covalent catalysis

Enzyme forms a covalent bond with the substrate

Carbonic anhydrases biological needs

Exhalation of CO2 in the lungs 2. Generation of aqueous humor in the eyes 3. Reabsorption of electrolytes by kidneys

A competitive inhibitor decreases the maximum rate at which a reaction can proceed. T/F

False

An active site histidine abstracts a proton from a substrate, making it more reactive, then donates the proton back to the product. What example of catalysis is this?

General acid/base catalysis

What forms the oxyanion hole of chymotrypsin?

Gly 193 and Ser 195

Nucleoside Monophosphate Kinases: P-Loop Structure

Has an amino acid sequence of the form Gly-X-X-X-X-Gly-Lys

Chymotrypsin mechanism

His 57 catalyzes removal of H from Ser 195 hydroxyl. Ser 195's nucleophilic O attacks carbonyl C of substrate. His 57 donates H to N of sissile peptide bond, tetrahedral intermediate decomposes. The portion (C-terminal end) of original substrate with the new amino terminus diffuses away. Water donates H to His 57. Resulting OH attacks carbonyl of remaining substrate. His 57 donates H to Ser 195 O, leading to collapse of tetrahedral intermediate. The portion (N-terminal end) of original substrate with the new carboxylate terminus diffuses away.

Chymotrypsin mechanism step 1

His 57 catalyzes the removal of hydrogen from Ser 195 hydroxyl group

Chymotrypsin mechanism step 7

His 57 donates H to Ser 195 O, leading to collapse of tetrahedral intermediate

Chymotrypsin mechanism step 3

His 57 donates hydrogen to nitrogen on the peptide bond, the tetrahedral intermediate decomposes

Catalysis by approximation

In cases of two distinct substrates, the reaction rate may be increased significantly by bringing the two substrates together along a single binding surface on an enzyme (Ex. NMP kinase bring two nucleotide together to facilitate the transfer of a phosphoryl group from one nucleotide to the other.)

Recognition sequences aka recognition sites

In the target DNA is recognized by these enzymes and cleaved as defined positions

HIV protease inhibitor

Indinavir, mimics the transition state

NMP kinase is an example of

Metal ion catalysis, the metal forms a complex with the substrate to enhance enzyme-substrate interactions

Metal ion catalysis

Metal ions function in a number of ways, including serving as an electrophilic catalyst

Restriction enzymes: methylation of adenine Recognition sites in the host DNA are modified by the addition of a methyl group, a reaction catalyzed by ________

Methylases

Multiple ____ are required for cofactors for restriction enzymes

Mg+2

ATP must be bound to _______ or _______ to function as a substrate for myosin, along with most enzymes that hydrolyze nucleoside triphosphates require the nucleotide to be in a complex with _______ or ______

Mg+2, Mn+2

Hydrogen bond link ATP to peptide _______ groups as well as a lysine residue

NH

catalytic triad of chymotrypsin: Asp102

Orients the histidine and renders it a better proton acceptor

Chymotrypsin mechanism step 2

Oxygen of hydroxyl group on serine 195 makes a nucleophilic attack of carbonyl carbon of the substrate

Nomenclautre for protease-substrate interactions The potential sites of interactions of the substrate with the enzyme are designated with a ____

P

Myosins are a family of enzymes containing __-_____ structures

P-loop

Restriction enzymes aka restriction endonucleases

Productive strategy for host bacteria from a viral infection to degrade the viral DNA on its introduction into a cell

catalytic triad of chymotrypsin: His57

Removes a proton from serine 195, generating a highly reactive alkoxide ion

Phosphatases

Removes phosphoryl groups from proteins Turn off signaling pathways triggered by kinases Regulate the activity of other proteins

Restriction enzymes and methylases are called

Restriction modification system

Chymotrypsin mechanism step 6

Resulting OH attacks carbonyl of remaining substrate

Nomenclautre for protease-substrate interactions The corresponding binding sites on the enzyme are designated with a ____

S

Substantial conformational changes that occur during catalysis of ATP

Small changes at the active site are amplified by large changes in the carboxyl terminal region of myosin

Competitive inhibitor

Structurally similar to substrate When present, Km of enzyme will increase Prevents substrate from binding enzyme

oxyanion hole in chymotrypsin

Structure that stabilizes the tetrahedral intermediate of the reaction, Hydrogen bonds link peptide NH groups and the negatively charged oxygen

The binding energy establishes

Substrate specificity

Chymotrypsin mechanism step 4

The C-terminal portion of the substrate with the new N-terminal diffuses away

The S1 pocket has a negative charge. Consider the charge of the amino acid likely to interact with this pocket.

The S1 pocket has a glutamic acid residue at the bottom. Glutamic acid is negatively charged at physiological pH, and therefore attracts a positively charged amino acid. Look for amino acids with positively charged side chains. (Histidine\'s R group may be neutral or positively charged near neutral pH, as its pKa is around 6. Lysine (Lys) or arginine (Arg), and possibly histidine (His), could fit in the S1 pocket.

The S1' pocket is deep and hydrophobic. Look for a bulky hydrophobic side chain for the corresponding amino acid.

The S1' pocket is deep and hydrophobic, and can accommodate bulky, hydrophobic side chains. Leucine (Leu), isoleucine (Ile), methionine (Met), tryptophan (Trp), or phenylalanine (Phe) could fit in the S1' pocket.

The S2 pocket is small and hydrophobic. Consider the size of the amino acid side chain that could fit in this pocket. Is the side chain more likely to be hydrophobic (nonpolar) or polar?

The S2 pocket is small and hydrophobic. Therefore, the amino acid side chain must be small and hydrophobic. A large side chain would not fit. Hydrophobic (nonpolar) residues are attracted to other hydrophobic residues. Glycine (Gly), alanine (Ala), or valine (Val) could fit in the S2 pocket.

Covalent Catalysis

The active site contains a nucleophile that is briefly covalently modified.

How does this zinc complex facilitate carbon dioxide hydration

The binding of water molecule to the positively charged zinc center reduces the pKa of the water molecule from 15.7 to 7 A zinc-bound hydroxide ion is nucleophilic to attack carbon dioxide much more readily than water does

Catalysis by approximation info

The enzyme brings two substrates together in an orientation that facilitates catalysis

N-Tosylamido-L-phenylethyl, also called tosyl phenylalanyl chloromethyl ketone, (TPCK) irreversibly inhibits chymotrypsin, and is used as to label the active site histidine residue. The COCH2Cl group is the reactive group that binds to the His residue. Why does chymotrypsin bind TPCK?

The phenyl group of TPCK is structurally similar to regular chymotrypsin substrates

Chymotrypsin mechanism step 8

The portion (N-terminal end) of original substrate with the new carboxylate terminus diffuses away

Deacylation

The removal of one or more acyl groups (-COR) from a compound

Kinases function

Transfer a phosphate from a nucleotide triphosphate to another substrate

A higher subtrate concentration is associated with a faster reaction rate if the enzyme is not saturated. T/F

True

When substrate is present in excess, the maximum reaction rate is unchanged in the presence of a competitive inhibitor. T/F

True

The interactions between the Mg+2 and the _______ atoms in the phosphoryl group hold the nucleoside in well-defined conformations

oxygen

Mg+2 neutralizes some of the negative charges present on the ________ chain, reducing nonspecific ionic interactions

phosphate

Methylation prevents hydrolysis by restriction enzymes by

preventing the formation of contacts necessary to distort the DNA

The challenge for NMP kinases

promote the transfer of the phosphoryl group from NTP to NMP without promoting the competing (NTP hydrolysis, transfer of a phosphoryl group from NTP to water)

The carbonyl carbon atom in the peptide bond is less electrophilic and less susceptible to nucleophilic attack, so to promote peptide bond cleave, the enzyme ________ must facilitate nucleophilic attack at a normally unreactive carbonyl group

proteases

How is DNA protected by restriction enzymes degrading host DNA

protection by methylation

Chymotrypsin turns _______ into a highly reactive residue, the nucleophile that attacks the carbonyl group of the protein substrate

serine 195

A water molecule, assisted by ____ ______ attacks the γ phosphate of ATP, resulting in hydrolysis

serine 236

Interactions between the enzyme and the substrate favor ________ _________ and also stabilize the transition state, thereby lowering the activation energy

substrate binding

Restriction enzymes themselves display a corresponding ________. The enzymes are dimers in which the subunits are related by twofold _________ (same word)

symmetry

catalysis

the acceleration of a chemical reaction by a catalyst

covalent catalysis

the active site contains a reactive site, a powerful nucleophile that is briefly covalently modified (Ex. The covalently bound enzyme-substrate intermediate are formed in chymotrypsin action)

Mg+2 helps to activate a _________ molecule and positions it so it can attack the phosphate

water

Although the hydrolysis is thermodynamically favored, hydrolysis is extremely slow in the absence of a _______

catalyst

The binding energy increases

catalytic efficiency

Kinases

catalyze phosphorylation reactions May use ATP as a phosphoryl group donor PKA (protein kinase A) is an example Regulate the activity of other proteins

Chymotrypsin catalytic triad function

converts serine 195 into a potent nucleophile

The structure of noncognate DNa is not distorted by the _________

enzyme

Mutations in any component of the catalytic triad cause a dramatic loss of ______ ______

enzyme activity

General acid catalysis

general acid donates a proton to substrate. - Enzyme active site residue must be protonated!

General base catalysis Example

general base accepts a proton from the substrate - Enzyme active site residue must be deprotonated!

Most restriction enzymes recognizes that are _________ _________, which display a twofold rotational symmetry

inverted repeats

Binding of the enzyme to the recognition sequence distorts the DNA by introducing a _____

kink

________ or _________ complexes of nucleoside triphosphates are the true substrates for these enzymes

magnesium, manganese

Other proteases: aspartyl protease

Use an aspartate-activated water molecule as a nucleophile

Other proteases: cysteine proteases

Uses a histidine-activated cysteine residue as a nucleophile

Other proteases: metalloprotease

Uses a metal-activated water molecule as a nucleophile

Chymotrypsin mechanism step 5

Water donates H to His 57

Acylation

When an acyl group (-COR) is added to a molecule

Uncompetitive inhibitor

When present, Km of enzyme will decrease, Vmax decreases Binds enzyme-substrate complex only

Carbonic anhydrases: Zinc-containing enzyme

Zinc is bound to the imidazole rings of three histidine residues as well as to a water molecule in human carbonic anhydrase II

Metal ion catalysis states

Zinc is only found in +2 state in biological systems Essentially always bound to four or more ligands

Chymotrypsin

a proteolytic enzyme (enzymes that break down proteins) secreted by the pancreas that hydrolyzes peptide bonds selectively on the carboxyl side of large hydrophobic amino acids Three polypeptides, forming one chain, connected by 3 disulfide bonds

Nucleoside Monophosphate Kinases: substrates The metal is not a component of the _____ ______

active site

NMP kinases are essentially inactive in the absence of divalent ions such as Mg2+ or Mn2+, but acquire ________ on the addition of these ions.

activity

HIV Protease

an aspartyl protease needed for the maturation of viral coat protein

The distorted DNA makes additional contacts with the enzyme, thereby increasing the _____ ________

binding energy

chymotrypsin specificity pocket

binds to aromatic amino acids, bulky nonpolar side chain