Finding a suitable proteinligand system

When we first began our work in this area, we identified four main criteria for a suitable system with which to probe protein-ligand interactions. These were:

1. Pure, native protein was available for assays and structure determination.

2. A reliable assay was available to measure binding affinity.

3. The structure of the protein-ligand complex was relatively straightforward to determine at high resolution.

4. A large number of ligands were available with subtly varying chemistry.

The enzyme penicillin acylase (penicillin amidohydrolase EC 1) satisfied all of these criteria. This enzyme catalyses the hydrolysis of the amide bond in benzylpenicillin (Penicillin G, 1, Scheme 1) to give phenyl acetic acid and 6-aminopenicillanic acid.

The first structure of the enzyme [1] revealed a well-defined binding cavity for phenylacetic acid (Figures la, b). A survey of chemical suppliers catalogues identified a number of phenylacetic acid derivatives and so we

Scissile amide bond

Scheme 1. Structure of Penicillin G, 1

Scissile amide bond

Scheme 1. Structure of Penicillin G, 1

determined the structure of the enzyme in complex with a set of these ligands [2] (see Table 1). A relatively straightforward enzyme assay was available and the inhibition constants measured could be used to reflect the binding affinities of the different ligands.

The apparent binding affinity was consistent with simplistic modelling of the different ligands into the original structure. However, the structures revealed that the protein undergoes a conformational change on ligand binding (Figure 1c).

Two distinct conformations are possible; therefore the affinity measured is the overall balance of energetics for the ligand binding to the enzyme and the energy penalty for inducing conformational change in the enzyme.

These experiments added another requirement to our criteria for an experimental system for measuring protein-ligand interactions:

5. The structure of the protein and orientation of the ligand remain constant across the series of complexes.

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