Abstract
The high reactivity of the serine residue at the active site of serine proteases is often attributed to the formation of a hydrogen bond between this serine and a histidine residue. In the case of the serine protease subtilisin, the catalytic serine residue can be specifically replaced by a cysteine residue and this modified enzyme is called thiol-subtilisin. By studying the D2O effect on acyl-enzyme formation with subtilisin and thiol-subtilisin, we present evidence that thiol-subtilisin but not subtilisin may contain a hydrogen bond. Based on the comparison of the catalytic activities of subtilisin and thiol-subtilisin, a rigid active site model for the serine proteases is proposed in which the histidine residue operates in a fixed steric position both as a general base and as a general acid, and this, rather than the formation of a hydrogen bond, accounts for the high nucleophilicity of the serine residue.
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