LJS Chemistry 474 Chapter 6 Lecture Notes PDF

CHEM 474/674 – Biochemistry I Chapter 6: Enzyme Catalytic Strategies 1 Enzymatic Challenges ◼ To speed up really slow reactions ◼ To achieve a high rate of reaction suitable for integration into other rapid physiological processes ◼ At...

CHEM 474/674 – Biochemistry I Chapter 6: Enzyme Catalytic Strategies 1 Enzymatic Challenges ◼ To speed up really slow reactions ◼ To achieve a high rate of reaction suitable for integration into other rapid physiological processes ◼ Attain a high degree of specificity ◼ Utilize free energy associated with the hydrolysis of ATP to drive other processes 2 Catalytic Strategies ◼ Covalent Catalysis ❑ The active site of the enzyme contains a reactive group (usually a nucleophile) that becomes temporarily covalently attached to the substrate ◼ What types of residues can act as nucleophiles? ❑ Unprotonated His imidazole ❑ Unprotonated -amino group ❑ Unprotonated -amino group of Lys ❑ Unprotonated thiol (thiolate anion, S-) of Cys ❑ Aliphatic –OH of Ser ❑ Unprotonated R group (carboxylates) of Glu, Asp 3 Catalytic Strategies ◼ General Acid-Base Catalysis ❑ A molecule other than water plays a role as a proton acceptor (base) or donor (acid) ❑ The group that donates a proton in catalysis has to accept a proton later in the mechanism to regenerate the enzyme (and vice versa) ◼ What kind of residues can act as general acids and bases? ❑ His imidazole ❑ -amino group ❑ Thiol of Cys ❑ R group carboxyls of Glu, Asp ❑ -amino group of Lys ❑ Aromatic OH of Tyr ❑ Guanidino group of Arg 4 Catalytic Strategies ◼ Catalysis by Approximation ❑ Most enzymes catalyze reactions between 2 substrates. These enzymes often have a single binding surface for both substrates, to bring them close together. ◼ Metal Ion Catalysis ❑ Metal ions can play several potential roles in enzymatic reactions ◼ May facilitate the formation of a nucleophile (by coordination) ◼ May serve as an electrophile, stabilizing negative charge on a reaction intermediate ◼ May serve as a bridge between an enzyme and a substrate 5 Enzyme Mechanisms ◼ Chymotrypsin ❑ Cleaves peptide bonds after bulky, aromatic residues ◼ Carbonic anhydrase ❑ Adds water to carbon dioxide ◼ Restriction endonuclease (EcoRV) ❑ Hydrolyzes/cleaves DNA at specific sites ◼ Myosin ❑ Hydrolyzes ATP 6 What makes a good nucleophile? ◼ “Wants” to give away electrons (a good Lewis base) 7 What makes a good nucleophile? ◼ More polarizable (bigger); not sterically hindered 8 What makes a good nucleophile? ◼ Not obscured by too polar a solvent 9 Enzyme 1: Chymotrypsin ◼ Digestive enzyme ◼ Synthesized in mammalian pancreas ◼ Involved in protein turnover ◼ Cleaves peptide bonds after large, bulky residues, like F, Y, and W 10 Chymotrypsin ◼ The problem: ❑ Chymotrypsin needs to speed up an incredibly slow reaction. ◼ The catalytic task: ❑ To make a normally unreactive carbonyl group more susceptible to nucleophilic attack by water ◼ The strategy: ❑ Covalent catalysis (assisted by general base catalysis) 11 Chymotrypsin: A Mechanism in Two Phases ◼ Acylation to form the acyl-enzyme intermediate ❑ The first substrate (protein/peptide) adds ❑ The Serine nucleophile attacks the carbonyl of the peptide bond to be cleaved ❑ The amine component of the bond is released (the C-terminus of the original protein, with a new amine group) ◼ Deacylation to release the carboxylic acid component of the substrate and regenerate the enzyme ❑ The second substrate (water) adds ❑ Nucleophilic attack by water on the carbonyl group of the original peptide bond ❑ Release of the carboxylic acid component of the bond (the N-terminus of the original protein, with a new carboxyl group) 12 Chymotrypsin Mechanism 13 Acyl Groups Serine 14 Chymotrypsin Structure ◼ Spherical ◼ Composed of 3 polypeptide chains 15 Chymotrypsin Active Site ◼ Cleft on the surface of the enzyme ◼ Catalytically important residues are Ser195, His57, and Asp102 ❑ “Catalytic Triad” 16 Chymotrypsin Mechanism Nucleophilic Collapse of the attack by Ser tetrahedral on carbonyl intermediate First substrate (protein) binds Release of the amino component Release of the carboxy component H2O binds Collapse of the Nucleophilic tetrahedral intermediate attack by water 17 on carbonyl Chymotrypsin Mechanism: Things to Remember ◼ The overall reaction is: protein + H2O peptide + peptide ◼ There are two general phases that are essentially repeats of each other but with different nucleophiles: ❑ Acylation phase (steps 1-4): activated Ser acts as a nucleophile, attacks the carbonyl in the peptide bond to be cleaved and releases the amine component of the bond ❑ Deacylation phase (steps 5-8): activated water acts as a nucleophile, attacks the carbonyl in the peptide bond to be cleaved, and releases the carboxyl component of the bond 18 Step 1: First Substrate Binds ◼ In the first step of the acylation phase, the first substrate (protein) binds ◼ Bound substrate is positioned so that the peptide bond on the C-terminal side of F, W, or Y can be cleaved 19 Step 2: Formation of First Tetrahedral Intermediate ◼ His57 catalyzes the removal of a proton from Ser195 to generate the alkoxide ion (His57 acts as a general base catalyst) ◼ The oxygen atom of Ser195 carries out a nucleophilic attack on the carbonyl carbon of the target peptide bond 20 Oxyanion Hole ◼ Nucleophilic attack of the Ser alkoxide group on the carbonyl of the peptide bond generates a tetrahedral intermediate with a negative charge on the oxygen ❑ The negative charge is stabilized by interactions with NH groups from the protein in a site termed the “oxyanion hole.” These interactions stabilize the transition state of this step of the reaction. 21 Step 3: Formation of Acyl-Enzyme Intermediate ◼ First tetrahedral intermediate breaks down -- original amide (peptide) bond cleaves ❑ HisH+ donates a proton to the amino "half" of the original substrate (HisH+ acts as general acid) to generate R2-NH2 (R2 is from carboxyl terminus of original peptide). ❑ Breaking of amide bond generates acyl-enzyme intermediate 22 Step 4: Amine Product Dissociates from Active Site (1st Product leaves) ◼ “Amine” product dissociates from active site ❑ “Amine” product is the C- terminus of the original peptide, with a new amine group (from the broken peptide bond) 23 Step 5: Binding of Second Substrate (H2O) in Active Site ◼ At the beginning of the deacylation phase, water takes the place formerly occupied by the amine component of the substrate 24 Step 6: Formation of Second Tetrahedral Intermediate ◼ His57 acts as a general base catalyst, drawing a proton away from H2O ◼ The resulting OH- carries out a nucleophilic attack on the carbonyl of the acyl group to form a tetrahedral intermediate ❑ The intermediate is stabilized in the oxyanion hole. 25 Step 7: Breakdown of Second Tetrahedral Intermediate ◼ HisH+ (general acid) donates proton back to the Ser O, generating the alcohol product of the hydrolysis of the acyl- enzyme, Ser-OH ◼ The ester bond from acyl-enzyme intermediate breaks to give the carboxylic acid product (R1-COOH) from the original substrate. 26 Step 8: Carboxylic Acid Product Dissociates from Active Site ◼ “Carboxylic Acid” product dissociates from active site ❑ “Carboxylic acid” product is the N-terminus of the original peptide, with a new carboxyl group (from the broken peptide bond) 27 Chymotrypsin Mechanism Nucleophilic Collapse of the attack by Ser tetrahedral on carbonyl intermediate First substrate (protein) binds Release of the amino component Release of the carboxy component H2O binds Collapse of the Nucleophilic tetrahedral intermediate attack by water 28 on carbonyl Questions! ◼ Does hydrolysis occur in the acylation or deacylation half-reaction of serine proteases? ◼ What is the nucleophile in the acylation half-reaction? ◼ What is the nucleophile in the deacylation half-reaction? 29 Specificity of Chymotrypsin ◼ Specificity is mostly due to the structure of its “specificity pocket” ❑ A pocket in the active site adjacent to the catalytic site ❑ In chymotrypsin, the specificity pocket (S) is deep, hydrophobic, and lined with small Gly residues. ❑ Binding of an appropriate side chain into this pocket positions adjacent peptide bonds into the active site for cleavage 30 Specificity Patterns ◼ While chymotrypsin only has one specificity pocket that is located in its active site, other enzymes have very complex specificity patterns that necessitate having multiple specificity pockets (some of which might be on the surface of the enzyme and not just in the active site). 31 Specificity Pockets ◼ Many other enzymes have catalytic triads ❑ Each of these cleaves bonds by similar mechanisms, but has a different specificity pocket Cuts on the C- Cuts on the C- Cuts on the C- terminus of large, terminus of residues terminus of bulky, aromatic with positively residues with residues charged side chains small side chains 32 Learning Check ◼ Thrombin is a protease that specifically cleaves Arg-Gly bonds. You would predict that A. The S1 site will contain an Asp, while the S1’ site will be large and hydrophobic B. The S1 site will contain an Asp, while the S1’ site will be small and shallow C. The S1 site will be large and hydrophobic while the S1’ site will be negatively charged D. The S1 site will contain a Lys, while the S1’ site will be large and hydrophobic E. None of the above 33 Enzyme 2: Carbonic Anhydrase CO2 + H2O H2CO3 HCO3− + H+ 34 Carbonic Anyhydrase ◼ The problem: ❑ Carbonic anhydrase needs to speed up a reaction to match the rates of other rapid physiological processes ◼ The catalytic tasks: ❑ To make water a stronger nucleophile and to find a way to move protons to the enzyme at a rate faster than the diffusion rate ◼ The strategy: ❑ Metal ion catalysis (for generating the strong nucleophile) 35  Structure of Carbonic Anhydrase Carbonic anhydrase requires a Zn2+ cofactor: Zinc coordinates to ligands in the protein and to a water molecule Binding of Zn2+ to water lowers water’s pKa, making it easier to deprotonate and generating a potent nucleophile 36 Binding Sites in Carbonic Anhydrase Active Site ◼ Next to the Zn2+ binding site is a hydrophobic patch that serves as a binding site for CO2 37  Carbonic Anhydrase Mechanism 1. Proton 2. Hydroxide ion released generated from water 6. Bicarbonate 3. CO2 binds released as another H2O binds 4. Nucleophilic attack 5. HO- attack converts CO2 to bicarbonate 38 Enzyme 3: Restriction Enzymes (EcoRV) ◼ Catalyze the hydrolytic cleavage of DNA ◼ Mechanism for protection against viral infections ◼ Recognize and cleave particular base sequences ❑ “Recognition sequences” or “recognition sites” 39 Restriction Enzymes ◼ The problem: ❑ Restriction enzymes need to cleave viral DNA at VERY specific sites ◼ The catalytic tasks: ❑ To (1) cleave only DNA containing the recognition sequence (“cognate DNA”), (2) cleave viral DNA and not host DNA, and (3) create a good nucleophile ◼ The strategies: ❑ Assembly of the complete catalytic apparatus only on binding of cognate DNA molecules, metal ion catalysis (and cofactors), and modification of host DNA 40 Reaction Catalyzed by Restriction Enzymes 41  Activating Water Restriction enzymes require Mg2+ cofactors ◼ May need as many as 3 magnesium ions per active site ◼ One ion-binding site is occupied by Mg2+ in essentially all restriction enzyme structures ❑ It is coordinated to 2 Asp residues and to one of the phosphate group oxygen atoms near the site of cleavage. ❑ This metal ion binds the water molecule that attacks the phosphorous atom, helping to position and activate it (like the zinc ion in carbonic anhydrase). 42 Specificity of Restriction Enzymes 43 Symmetry of Restriction Enzymes ◼ The two-fold symmetry of cognate DNA is matched by the two-fold symmetry of restriction enzymes 44 How specific is DNA-restriction enzyme binding? ◼ It seems like restriction enzymes should only bind to the sequences they cleave, but binding studies in the absence of magnesium ions show that restriction enzymes bind to all sequences—specific and nonspecific—with approximately equal affinity. 45  Post-binding Interactions ◼ In EcoRV, the 5′ end of GATATC hydrogen- bonds with residues in the enzyme 46 DNA Distortion ◼ The formation of the hydrogen bonds results in a distortion of the dsDNA in cognate DNA 47 Specific v. Nonspecific DNA ◼ DNA without this specific DNA sequence is not substantially distorted (the weak interactions don’t line up to form hydrogen bonds that result in the distortion of the DNA) ❑ As a consequence, the phosphodiester bonds in nonspecific DNA are not close enough to complete the magnesium ion binding site that is necessary for cleavage In the case of restriction enzymes, specificity is determined by the specificity of enzyme action (how it interacts with bound molecules) 48 instead of by the specificity of enzyme binding. Protecting the Host DNA through Modification ◼ The host cell has enzymes called methylases that add methyl groups to certain bases in the host DNA ❑ Methylation protects the host DNA from hydrolysis by the restriction enzymes 49  Methyl Group Protection ◼ The methyl group interferes with the formation of the hydrogen bond between the DNA and the enzyme ◼ As a result, the DNA is not distorted ◼ The presence of the methyl group protects the host DNA from hydrolysis ❑ Since the viral DNA is not methylated, it can be cleaved. 50  Dianne Beatty ◼ As a diabetic, Di Beatty injects herself several times a day as part of an insulin replacement regime. Insulin promotes glucose utilization as a fuel and glucose storage as fat and glycogen. Insulin is also an important regulator of hexokinase. Hexokinase is a bisubstrate enzyme that catalyzes the reversible reaction shown to the right: 51 Dianne Beatty ◼ ATP and ADP always bind to enzymes as a complex with the metal ion Mg2+. The hydroxyl at C-6 of glucose (to which the γ- phosphoryl group of ATP is transferred in the hexokinase reaction) is similar in chemical reactivity to water, and water freely enters the enzyme active site. Yet hexokinase favors the reaction with glucose by a factor of 106. The enzyme can discriminate between glucose and water because of a conformational change in the enzyme when the correct substrates bind. 52 Dianne Beatty Before Substrate Binding After Substrate Binding ◼ Why would this conformational change allow for the substrate discrimination? 53 Enzyme 4: Myosins ◼ Enzymes that catalyze the hydrolysis of ATP to ADP and Pi ❑ They use the energy associated with this thermodynamically favorable reaction to drive the motion of molecules within cells 54 Myosins ◼ The problem: ❑ Myosins need to use the free energy associated with the hydrolysis of adenosine triphosphate to drive other processes (usually associated with movement) ◼ The catalytic tasks: ❑ To activate the nucleophile, hydrolyze ATP in a controlled manner and use the energy to promote conformational changes in the myosin molecule in order to transport proteins or other cargo within the cell ◼ The strategies: ❑ Step-wise conformational changes in enzyme, general base catalysis (assisted by a metal ion) 55 Myosin Hydrolysis of ATP ◼ Occurs by nucleophilic attack of activated water on the -phosphoryl group of ATP 56  Structure of Myosin ATPase Domain ◼ Single globular domain with a water- filled pocket that includes a Enzyme is inactive in the absence of either nucleotide Mg2+ or Mn2+, but the metal ion is NOT a component of the active site (ATP binds the binding site metal ion before it binds to the enzyme). The metal-nucleotide complex is the TRUE substrate for this enzyme. 57 Myosin Mechanism ◼ Before the water can attack the -phosphoryl group of ATP, it must be activated, but how? ❑ With a basic residue? ◼ There are no basic residues in an appropriate position to activate the water when the substrate initially binds to the enzyme. ❑ With a metal ion? ◼ The metal ion is also too far away when the substrate initially binds to the enzyme. ◼ Therefore, the enzyme must undergo a conformational change to catalyze the reaction. 58 Conformational Changes in Myosins ◼ The enzyme goes through a series of conformational changes on binding and the subsequent hydrolysis of ATP. ❑ Around the active site, some residues move. A stretch of amino acids moves closer to the nucleotide by ~2 Angstroms. ❑ These amino acids interact with the attacking water molecule. ❑ This change facilitates the hydrolysis reaction by stabilizing the transition state. 59 Myosin Mechanism ◼ Mg2+ is coordinated to two phosphate groups of ATP, two hydroxyl groups from the enzyme, and two water molecules. ❑ The Mg2+ ion binds the substrate in a particular conformation. ❑ In this position, Ser236 is well-positioned to play a role in catalysis ◼ Water attacks the -phosphorous with the –OH of Ser236, facilitating transfer of a proton from the attacking water to the –OH of Ser236, which, in turn, is deprotonated by one of the O atoms of the -phosphoryl group. ❑ ATP serves as the general base to promote its own hydrolysis through the activation of the attacking water molecule. 60 Further Conformational Changes ◼ As the substrate changes into the transition state and as the reaction continues, a region of about 60 amino acids at the C- terminus of the protein (the enzyme) is displaced by as much as 25 Å from its original position 61  Dianne Beatty ◼ Induced fit is only one aspect of the catalytic mechanism of hexokinase. Like chymotrypsin, hexokinase uses several catalytic strategies. For example, the active site amino- acid residues (those brought into position by the conformational change that follows substrate binding) participate in general acid-base catalysis and transition-state stabilization. 62 Dianne Beatty ◼ The active site of hexokinase contains five residues: Lys-169, Thr- 168, Asn-204, Asp-205, and Glu-256. Asp-205 acts as the general base that abstracts a H from the the –OH group on D- glucose’s C6. Draw the mechanism of glucose phosphorylation. 63 Dianne Beatty 64 Dianne Beatty 65 Dianne Beatty 66 Dianne Beatty 67