Substrate analogues: checking the role of functional groups

1

• Substrate analogues checking the role of
  functional groups
• Identification of intermediates in the reaction
  path
• Wrong substrates undergo unexpected side
  reactions
• Inhibitors form stable intermediates or stable
  covalent side products
• Stereochemistry of the substrate and product
  discern possible paths
• Trapping of intermediates direct visualization

2

• Substrate analogues

H
3

• Substrate analogues

Loss of the S (thioester) does not affect binding
(and radical formation and catalysis), but loss
of the carbonyl group does.
4

• Substrate analogues

Loss of the S (thioester) does not affect binding
(and radical formation and catalysis), but loss
of the carbonyl group does. The carbonyl group
binds tightly and provides the energy.
5

• Wrong substrates bromopyruvate with
  transaminase

Inhibition by covalent modification
1.
2.
non-enzymatic
6

• Wrong substrates bromopyruvate with
  transaminase

wrong reaction that explains the product
pyruvate
make quinone
stabilize tetrahedral intermediate
unexpected reaction
7

• Inhibitors Kinetic analysis shows, that another
  observed pathway (to the covalently inhibited
  enzyme) must have the same intermediate

The base can react with the common intermediate,
an enamine.
8

• Stereochemistry of the substrate and product

9

• Stereochemistry of the substrate and product

10

• Stereochemistry of the substrate and product

L-haloacid dehalogenase direct attack by H2O or
via a covalent intermediate?
11

• Trapping of intermediates

L-haloacid dehalogenase has a pH optimum of 9.5.
At pH 5 the reaction is trapped at an
intermediate. This stable intermediate can be
characterized in a crystal structure.
12

• Trapping of intermediates

13

• Trapping of intermediates
• Why is the intermediate trapped at pH 5?
• In the structure at pH 5, no water molecule is
  close to Asp 8 and available for hydrolysis.
• At pH 9.5 (the pH optimum) a structural change
  must occur and a water molecule enters the active
  site. Moreover, at higher pH, water is more
  likely to deprotonate and then more nucleophilic.