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Interdependency of the binding subsites in subtilisin.

H Grøn¹, K Breddam

¹Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark.

|September 22, 1992

Summary

Substrate interactions with subtilisin enzymes like Savinase are complex and interdependent, not always additive. Optimizing substrates requires considering these interactions to achieve maximum catalytic efficiency (kcat/KM).

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Area of Science:

Biochemistry
Enzymology
Protein Science

Background:

Subtilisins are endopeptidases with extensive binding clefts (at least eight subsites).
Subsite interactions distant from the scissile bond significantly influence substrate specificity.
Understanding these interactions is crucial for enzyme engineering and drug design.

Purpose of the Study:

To investigate the interdependency of substrate-enzyme interactions in the subtilisin Savinase.
To determine how individual subsite contributions affect catalytic efficiency (kcat/KM).
To establish limits on substrate optimization for endopeptidases.

Main Methods:

Kinetic studies using a series of substrates designed to probe subsites S6 to S'4 of Savinase.
Analysis of the additive or non-additive contributions of individual subsite-substrate interactions to kcat/KM.
Evaluation of substrate-derived inhibitors.

Main Results:

Substrate-subsite interactions are not always additive, with remote subsites showing interdependency.
Favorable amino acids at P1 and P4 positions can compensate for unfavorable residues elsewhere.
An upper limit for kcat/KM (approx. 2 x 10^9 min-1 M-1) was observed, indicating a ceiling for transition-state stabilization.

Conclusions:

Enzyme-substrate interactions are complex and interdependent, particularly in subtilisins.
Optimal substrate or inhibitor design requires mapping these interdependent interactions.
Eliminating interdependencies in subsite mappings is key for designing effective endopeptidase substrates or inhibitors.

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