Hydrolases catalyze the cleavage of a bond by water:

1

• Hydrolases catalyze the cleavage of a bond by
  water
• Subclasses Which bond is hydrolyzed?

2

• Essential groups amino acid side chains,
  non-covalent intermediates
• Asp amidohydrolases
• Glu amidohydrolases (metalloproteases)

3

• Asp aspartic or acid proteinases (pepsin, HIV
  protease)

4

• Asp aspartic or acid proteinases (pepsin, HIV
  protease)

5

• Asp aspartic or acid proteinases (pepsin, HIV
  protease)
• The pH optimum is acidic for most aspartic
  proteinases, where one proton is bound by the two
  active site aspartates.
• Water is activated by the aspartates to undergo
  the nucleophilic attack.

6

• Asp aspartic or acid proteinases (pepsin, HIV
  protease)
• LBHB low barrier hydrogen bond

7

• Essential groups amino acid side chains,
  covalent intermediates
• Ser, Thr, Cys amidohydrolases
• Ser lactamases (class A, B, D)
• Ser esterases, phospholipases
• Asp dehalogenases

8

• Ser Serine proteases
• Aspects of the serine protease chymotrypsin
• Mechanism of action Acylation
• Anion hole
• Mechanism of action Deacylation

Nomenclature by Schechter.
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• Intermediates and transition states

E
Itetr
aspartate proteases
reaction
E
Itetr
Itetr
serine proteases
Iacyl
reaction
13

• Cys cysteine or sulfhydryl proteases (papain,
  cathepsins, picornain 3C)
• The active site contains a Cys-His dyad.

intermediate
14

• Cys cysteine or sulfhydryl proteases (papain,
  cathepsins, picornain 3C)

Triads and dyads All three (both) residues are
essential. They are connected by hydrogen bonds.
Although the Asp-His bond is strong, the proton
is not transferred to Asp.
15

• Thr Asparaginase
• The active site contains a Thr-Tyr dyad and a
  Thr-Lys-Asp triad.
• Both Thr--gtVal mutants are inactive.

16

• Thr Asparaginase
• The dyad catalyzes the acylation, the triad the
  deacylation.

17

• Thr Asparaginase
• The dyad Thr catalyzes the acylation and forms a
  covalent intermediate,
• the triad probably catalyzes the deacylation.

18

• Asp L-haloacid dehalogenase
• Note the stabilization of the leaving group Cl-
  by Arg and the stabilization of the tetrahedral
  intermediate by the anion hole.

19

• Asp L-haloacid dehalogenase
• Activation of the aspartate
• Asp-Lys-Asp

20

• Asp Lysozyme
• The reaction proceeds through a covalent
  intermediate, as with all b-glucosidases.

21

• Essential groups metal ions
• Zn2 amidohydrolases, lactamases (class B)
• Zn2, Mg2 phosphatases
• Fe2 arginase
• Ni2, V2 urease

22

• Zn2 Carboxypeptidase A (Stryer, old German
  edition, 1990)

?
23

• Zn2 Thermolysin
• without ligand with inhibitor
  (Phosphoramidon)

24

• Zn2 Thermolysin

Zn2 binds the amide carbonyl group and polarizes
the carbon. The nucleophile water is activated by
Glu143. His231 is the acid for protonating the
leaving group.
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• Zn2, Mg2 Alkaline phosphatases
• ROPO32- H2O ---gt ROH HPO42-
• Useful substrate p-Nitrophenylphosphate

26

• Zn2, Mg2, Ser
• Alkaline phosphatase
• The metal ions decrease the pKa of coordinated
  water molecules (Zn1 in E-P, Mg in E and E-P) and
  favor nucleophilic attack on coordinated
  phosphate (Zn1 in EROP).