• This leads to a tetrahedral intermediate and then an acyl enzyme, just as in the serine proteases

• The difference, which is small, is revealed by examination of the pH dependence of the enzyme activity:

  pH Dependence of Papain Activity

Such a "bell-shaped" curve is typical for many enzymes. The general implication:

• Activity requires two ionizable residues

• At low pH, the one that needs to be deprotonated is protonated; activity is minimal

• At high pH, the one that needs to be protonated is deprotonated, again minimizing activity

• At intermediate pH, sufficient enzyme molecules have both residues in the proper state of protonation, and catalysis can occur.

Explaining the pH Dependence of Papain

The mechanism is identical to that of the serine proteases:

• Note that no role is proposed for the Asn182 of actinidin; if it is to help, it must undergo a conformational change

• The Asn175 of papain could help, but is much less basic than an Asp.

• The residues comprising the "oxyanion hole" have not been specifically identified.

Here is an attempt to observe the tetrahedral intermediate:

Papain with AcLeuLeuArg-CHO Bound (1pop)

In this case, the enzyme has been fooled into forming a hemithioacetal, a covalent tetrahedral intermediate, by attacking the aldehyde carbonyl.

Papain is synthesized witha 107 amino acid "prosegment" that serves to inactivate it

• Little is know about the prosegment other than its sequence

• Activation occurs by a pH dependent autophagy, followed by attack of the activated enzyme on other prosegments