Evidence has been developed about the tetrahedral intermediate by the same stunt as for the Zn proteases: feeding the enzyme a phosphonate analog of the transition state as an inhibitor.

Human Pepsin Bound to Transition State Inhibitor	Close-up of Catalytic Site

The inhibitor was chosen not just for the phosphonate mimic of the tetrahedral intermediate, but also by analogy to a naturally occuring inhibitor of aspartate proteases called pepstatin:

Human Pepsin Bound to Pepstatin	Close-up of Catalytic Site

Note that both the natural and the synthetic inhibitors bind more closely to Asp32 than to Asp215; this is closer to the second mechanism than the first.

Pepsin is synthesized as a zymogen, which (as noted) is stable at normal physiological pH.

• When secreted into the stomach, at pH 3.5 or lower, it immediately undergoes autocatalytic cleavage of the 44-residue prosegment, producing active pepsin.

• As with other proteases employing a long prosegment, the activation peptide trails through the active site of the enzyme, providing a physical block:

Porcine Pepsin Zymogen (2psg)	Closeup of Catalytic Site

Note that the side-chain of the Lys36 of the prosegment extends down into the catalytic center, forming a salt bridge with the catalytic aspartates.

Pepsin has yet another trick up its binding cleft. In the proenzyme, residues 1-11 of the main chain also block the active site. When the prosegment is cleaved, these residues move away:

Motion of Residues 1-11 of Pepsin on Activation

In this superposition of the porcine pepsin with the main chain of its proenzyme:

• The green sequence marks the position of residues 1-11 in the zymogen

• The cyan sequence shows those residues in the active enzyme

• The CPK models are the catalytic aspartates