Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction.acyl-enzyme ͉ enzyme mechanism ͉ steady state ͉ reaction trajectory S erine proteases catalyze peptide bond hydrolysis in two sequential steps. In the first (acylation) reaction, the nucleophilic serine attacks the substrate scissile bond, forming first a tetrahedral intermediate and then a covalent acyl-enzyme with release of the C-terminal fragment. In the second (deacylation) reaction, a water molecule attacks the acyl-enzyme, leading to a second tetrahedral intermediate followed by release of the N-terminal fragment. This mechanism has long served as one of the classic paradigms illustrating the catalytic power of enzymes (1), yet crucial details remain controversial.It is generally accepted that a histidine residue acts as a general base in accepting a proton to activate serine as a nucleophile, and subsequently acts as a general acid, donating the proton to the nitrogen of the peptide leaving group (1). This same histidine is also presumed to deprotonate the hydrolytic water. What is not clear is why, if the histidine is ideally situated to deprotonate serine, does the first tetrahedral intermediate not collapse back to the reactant complex with return of the proton to serine? Does the active site undergo spatial reorganization as catalysis proceeds to favor the forward progress of the reaction (2, 3)? A reaction-driven ''His flip'' mechanism has been proposed to address this question (4) but has met with resistance (5-8).Another longstanding question concerns the positioning and activation of the hydrolytic water molecule and the trajectory of attack in the deacylation reaction (9-13).These questions can now be approached through the study of protein structures of intermediates along the catalytic pathway. Structures of the enzyme͞substrate Michaelis complex, analogs of the tetrahedral intermediates, and acyl-enzymes can yield great insight into the atomic motions and shifting geometric relationships along the reaction coordinate. Here, we report (i) high resolution structures of trypsin acylated by two good peptide substrates and (ii) higher resolution structures of two previously solved trypsin complexes: the stable acyl-enzyme formed with the poor substrate p-nitroguanidinobenzoate (14) and the covalent complex formed with the inhibitor leupeptin, which mimics a tetrahedral intermediate (15). Comparisons provide insight into active site adjustments involved in catalysis and cl...

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