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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.

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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-.

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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.

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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.

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Step 7

•  The tetrahedral intermediate breaks down.

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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.

•  This is how hydrolysis is prevented.

4. NMP kinases

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P-loop conservation 4. NMP kinases

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P-loop conservation cont. 4. NMP kinases