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S 5. This helix is nearer to the active site in renins than in other aspartic proteinases. On an equivalent
loop in the C-lobe (related by the intramolecular pseudo 2-fold axis), there is a sequence of three
prolines—the Pro292–Pro293– Pro 294 segment. This structure is also unique to the renins among the
aspartic proteinases with Pro294 and Pro297 in a cis configuration. Such a proline-rich structure
provides an effective means of constructing well-defined pockets from loops that would otherwise be
more flexible.
This rather rigid poly-proline loop, together with the loop comprised of residues 241–250, lies on either
side of the active site “flap” formed by residues 72–81. Hence, in the renins, the cleft is covered by the
flaps from both lobes rather than from the N-lobe alone as in other pepsin-like aspartic proteinases. This
gives renin a superficial similarity to the dimeric, retroviral proteinases where each subunit provides an
equivalent flap that closes down on top of the inhibitor [44,45].
B. The Role of Hydrogen Bonds in Inhibitor Recognition
Whereas the mouse renin inhibitor extends from P 6 to P 4', the human renin inhibitor extends only from
P 4 to P 1'. The cyclohexyl norstatine residue at P 1 in the human renin inhibitor mimics a dipeptide analog
with its isopropyloxy group occupying the subsite for the side chain of P 1'. The mouse renin inhibitor
(CH-66) possesses a Leu-Leu hydroxyethylene transition state analog [12]. Both inhibitors are bound in
the extended conformation that is found in other aspartic proteinase-inhibitor complexes. Both inhibitors
make extensive hydrogen bonds with the enzymes as shown in Figure 4. In general the two renin-
inhibitor complexes described here demonstrate that a similar pattern of hydrogen bonding is probably
used in the substrate recognition of all aspartic proteinases although their specificities differ
substantially.
There is also great similarity between aspartic proteinases in terms of interactions with the transitionstate
analog inhibitors at the catalytic center. The catalytic aspartyl side chains and the inhibitor
hydroxyl group are essentially superimposable in both renin complexes. The isostere C-OH bonds lie at
identical positions when the structures of inhibitor complexes of several aspartic proteinases are
superposed, in spite of the differences in the sequence and secondary structure. Most of the complex
array of hydrogen bonds found in endothiapepsin complexes are formed in renin with the exception of
that to the threonine or serine at 218, which is replaced by alanine in human renin. The similarity can be
extended to all other pepsin-like aspartic proteinases and even to the retroviral proteinases [44,45]. This
implies that the recognition of the transition state is conserved in evolution, and the mechanisms of this
divergent group of proteinases must be very similar.
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