10/4/10 1 1 Catalysis 2 4 Examples of enzymes • Adding water to a substrate: – Serine proteases. – Carbonic anhydrase. – Restrictions Endonuclease. • Transfer of a Phosphoryl group from ATP to a nucleotide. – Nucleoside monophosphate (NMP) kinase. 3 4 different challenges • Serine proteases - chymotrypsin: promoting a reaction that is immeasurably slow at neutral pH. • Carbonic anhydrase: Making a fast reaction even faster. • Restrictions Endonucleases - EcoRV: attaining a high level of specificity. • NMP kinase: Transfer of a Phosphoryl group from ATP to a nucleotide and not to water.
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Catalysis
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4 Examples of enzymes
• Adding water to a substrate: – Serine proteases. – Carbonic anhydrase. – Restrictions Endonuclease.
• Transfer of a Phosphoryl group from ATP to a nucleotide. – Nucleoside monophosphate (NMP) kinase.
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4 different challenges • Serine proteases - chymotrypsin: promoting a reaction that
is immeasurably slow at neutral pH. • Carbonic anhydrase: Making a fast reaction even faster. • Restrictions Endonucleases - EcoRV: attaining a high level
of specificity. • NMP kinase: Transfer of a Phosphoryl group from ATP to
a nucleotide and not to water.
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4 strategies for catalysis
1. Covalent catalysis. 2. General acid-base catalysis. 3. Metal ion catalysis. 4. Catalysis by approximation.
• They are not mutually exclusive!
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1. Covalent catalysis
• The active site usually contains a powerful nucleophile.
• The nucleophile is temporarily covalently bound to the substrate.
• Chymotrypsin is a good example.
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2. General acid-base catalysis
• An acid or a base plays the role of a proton donor or acceptor.
• Not water. • Again chymotrypsin’s active site is a good
example
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3. Metal ion catalysis
• Metals are good electrophilic catalysts stabilizing negative charges.
• They can also generate a nucleophile by increasing the acidity of an adjacent molecule (e.g. Carbonic anhydrase).
• The metal may bind the substrate to increase the binding energy (e.g. NMP kinase).
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4. Catalysis by approximation
• Bringing two substrates close together. • NMP kinase brings two nucleotides close
together so that the transfer of the Phosphoryl group is from one to the other.
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Proteases
• Proteins must have a certain turnover rate. • Many regulatory steps are achieved by the
concerted breakdown of proteins (e.g. cell cycle).
• Unfolded proteins are also degraded, so as not to cause any problems.
• In the gut proteins are broken down to their amino acid components.
1. Proteases
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The proteolytic reaction
• Addition of water to the peptide bond.
• The reaction is thermodynamically favored. • In the absence of a catalysis at neutral pH
however, t1/2 may be as long as hundreds of years.
RC
NH
R'
O
CRO
OR' NH3++ H2O +
1. Proteases
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Chymotrypsin • Chymotrypsin cleaves peptide bonds on the C-
terminal side of large hydrophobics.
• It is a good example of covalent modification as a catalytic strategy.
1. Proteases
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What is the nucleophile?
• Reactions with organofluorophosphates (e.g. DIPF) selectively labels Ser 195.
1. Proteases
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Monitoring kinetics
1. Proteases
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Kinetic analysis
1. Proteases
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A reaction in two stages A. Acyl enzyme intermediate formation releasing the
amine. B. Hydrolysis of the acyl enzyme releasing the COO-.
1. Proteases
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1. Proteases
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1. Proteases
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1. Proteases
The catalytic triad
• Asp 102 increases the catalytic power of H57. • His 57 serves as a general base catalyst. • Thus an alkoxide is formed which is a much
stronger nucleophile than a hydroxyl.
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The reaction as a whole
1. Proteases
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1. Proteases
Step 1
• Substrate binds. • Nucleophilic attack of
the alkoxide on the peptide carbonyl carbon.
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Step 2
• A change in the geometry of the peptide bond from trigonal planer to tetrahedral.
1. Proteases
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Step 2 cont.
• The formal negative charge on the carbonyl oxygen is stabilized by the oxyanion hole.
1. Proteases
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Step 3
• Collapse to an acyl enzyme intermediate.
1. Proteases
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Step 4
• The amine group leaves the enzyme.
• Thus half of the substrate remains bound to the enzyme.
1. Proteases
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Step 5
• A water molecules replaces the amine.
• His 57 acts as a general base catalyst again activating the water molecule.
• It now undertakes a nucleophilic attack on the acyl carbon.
1. Proteases
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Step 6
• Formation of an unstable tetrahedral intermediate.
1. Proteases
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Step 7
• The tetrahedral intermediate breaks down.
1. Proteases
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Step 8
• Release of the carboxylic acid.
• The cycle is now complete.
1. Proteases
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Specificity cause • A hydrophobic pocket selectively
binds large hydrophobic amino acids.
• Trypsin and elastase contain other pockets defining their specificity.
1. Proteases
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1. Proteases
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1. Proteases
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Different serine proteases: 1. Subtilisin
1. Proteases
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Different serine proteases: 2. Carboxypeptidase II
• Thus, the catalytic triad has appeared at least 3 times during the course of evolution!
1. Proteases
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Carbonic anhydrase (CA)
• CA catalyses the hydration and dehydration of CO2.
2. Carbonic anhydrase
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CA’s importance
• The natural rate of the reactions is fast: k1 = 0.15 s-1, however it is not fast enough.
• In the presence of the enzyme kcat = 106 s-1. • The need for the enzyme arises from the
fact that at times we need CO2 (e.g. in the lungs) and at time bicarbonate.
2. Carbonic anhydrase
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CA and Zinc
• CA was the first enzyme known to contain Zinc.
• Now as much as 1/3 of all enzymes are known to contain bound metal ions.
• Zinc is found in biology only as Zn2+. • It is normally coordinated by four ligands. • Remember that coordination is when one of
the partners in the bond donates the pair of electrons entirely.
2. Carbonic anhydrase
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• Due to the coordination the net charge due to the Zn2+ is 2+.
2. Carbonic anhydrase
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Catalysis and pH 1
.8
.6
.4
.2
k cat(1
06 s-1
)
• The midpoint in the transition is around pH 7.
• Thus a group with a pKA of 7 is critical to the enzyme’s activity.
• It is not a Histidine but rather a water molecule.
2. Carbonic anhydrase
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• Thus the binding of water to Zn2+ lowers the water’s pKA from 15.4 to 7.
2. Carbonic anhydrase
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The mechanism
2. Carbonic anhydrase
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Step 1
• Zn2+ facilitates the release of a H+ from the bound water molecule.
2. Carbonic anhydrase
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Step 2
• The CO2 binds in the enzyme’s active site.
• It is positioned accordingly for the attack.
2. Carbonic anhydrase
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Step 3
• Nucleophilic attack by the hydroxide ion. • The CO2 is converted to bicarbonate ion.
2. Carbonic anhydrase
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Step 4
• Regeneration of the catalytic site though the exchange of water and the release of bicarbonate.
2. Carbonic anhydrase
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The proton paradox • One of the steps in the reaction involves the deprotonation
of the water to form a hydroxide ion. • When the enzyme is working in the opposite direction
(dehydration of bicarbonate) the hydroxide ion protonates to form water.
2. Carbonic anhydrase
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The proton paradox cont. • Proton diffusion in water is very rapid, with second order rate
constants of 10 -11 M-1s-1. • Thus k-1 must be lower than 10 11 M-1s-1. • The equilibrium constant for H+ release,���
K = k1/k-1=10-7 M. • Thus k1 must be equal to 104 s-1. • In other words, the rate of H+ diffusion limits the rate of H+ release to
less than 104 s-1 for a group with a pKa= 7.
2. Carbonic anhydrase
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The proton paradox cont.
• However if CO2 is hydrated at a rate constant of 106 s-1 then every step in the reaction must proceed at least as fast.
• How can this be if the rate of proton release is only 104 s-1?
• How can this apparent paradox be resolved?
2. Carbonic anhydrase
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The proton shuffle
• The resolution of the paradox was possible upon noticing that maximal acceleration of the hydration reaction was only possible in the presence of buffer.
• The reason is that the [H+] is only 10-7M, but the concentration of the buffer can be much higher.
2. Carbonic anhydrase
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The proton shuffle cont.
• If the buffer (BH+) has a pKA of 7 (similar to the water molecule bound to the Zn2+) then the following equilibrium constant is obtained:
2. Carbonic anhydrase
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The proton shuffle cont.
• Now the rate of deprotonation k1’ (or the rate of H+ abstraction by the buffer) will be equal to: k1’ [B].
• The second order rate constants k1’ and k-1’ will be limited by buffer diffusion to values less than 109 M-1 s-1.
• Thus, [B] higher than 10-3 M will be able to support rate constant for hydration of CO2 of 106 s-1.
• This is because:���k1’ [B] = (109 M-1 s-1) (10-3 M) = 106 s-1.
2. Carbonic anhydrase
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The proton shuffle cont.
• Experimental date supports this prediction.
2. Carbonic anhydrase
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So what is the buffer?
• Most buffers are too big to reach the active site of the enzyme.
• For this reason the enzyme has positioned a His residue to act as the buffer in close proximity: a built-in H+ shuffle.
2. Carbonic anhydrase
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A built-in proton shuffle
• So the enzyme has evolved a mechanism to control H+ release and uptake to dramatically accelerate the rate of the reaction.
• This is seen in many other instances in which enzymes use acid-base catalysis.
• It also explains the prominence of such catalytic mechanisms.
2. Carbonic anhydrase
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Evolution of Zn2+ active sites
• The enzymes referred to so far are called α-carbonic anhydrases (α-CAs).
• Bacteria and pants contain β-CAs that are distinct from α-CAs, although they contain Zn2+ in their active site.
• The ligands for Zn2+ are 1 His and 2 Cys residues, as opposed to 3 His in α-CAs.
2. Carbonic anhydrase
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Nucleotide monophosphate kinases
• Nucleotide monophosphate (NMP) kinases catalyze the reversible transfer of a Phosphoryl from an NTP to an NMP.
• They can also be used to generate some NTP from two NDPs when NTP concentrations is being exhausted.
• Remember that: [ATP] > [ADP] > [AMP]
4. NMP kinases
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4. NMP kinases
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Adenylate kinase
• We will concentrate on adenylate kinase. • Its biggest challenge is to transfer the
Phosphoryl group to an AMP and avoid the competing reaction - hydrolysis.
• It provides an example for: – Induced fit. – Metal ion catalysis which is different than the
one used by the other enzymes previously discussed.
4. NMP kinases
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NMP kinases form a family 4. NMP kinases
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A core domain of an NMP kinase 4. NMP kinases
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The P-loop: G-XXXX-G-K 4. NMP kinases
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What is the real substrate? • The affinity of NTPs for Mg2+ (or Mn2+) is
10-4 M. • Since [Mg2+]~10-3 in the cell all NTPs are
found as: NTP-Mg2+
4. NMP kinases
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How does it affect catalysis?
• Mg2+ neutralizes the charge on the NTP to minimize non-specific interactions.
• The interactions between the Mg2+ and the NTP hold it in a stable conformation ready for catalysis.
• It provides for additional possibilities for interaction with the enzyme thereby increasing the binding energy.
4. NMP kinases
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• In some enzymes the Mg2+ is bound directly to the side chains (often E or D).
• In other there are bridging water molecules.
4. NMP kinases
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Binding induces a big conformational change
• The binding of ATP causes a large conformational change in the protein.
• The P-loop closes down on the ATP interacting with the β-phosphate.
• The movement of the P-loop enables the top domain of the protein to move closing down on the substrate further.
4. NMP kinases
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Catalysis
• Once the ATP is bound its γ-phosphate ions are positioned exactly near the AMP ready for catalysis.
• Binding of the AMP causes additional conformational change in the protein.
• Without the binding of both substrates the reaction will not take place.