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The molecular structures of three phosphorus-based peptide inhibitors of
aspartyl proteinases complexed with penicillopepsin [1, Iva-L-Val-L-Val-StaPOEt
[Iva = isovaleryl, StaP = the phosphinic acid analogue of statine
[(S)-4-amino-(S)-3-hydroxy-6-methylheptanoic acid] (IvaVVStaPOEt)]; 2,
Iva-L-Val-L-Val-L-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid analogue of
L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = methyl ester] [Iva
VVLP(O)FOMe]; and 3, Cbz-L-Ala-L-Ala-L-LeuP-(O)-Phe-OMe (Cbz =
benzyloxycarbonyl) [CbzAALP(O)FOMe]] have been determined by X-ray
crystallography and refined to crystallographic agreement factors, R ( = sigma
parallel to F0 magnitude of - Fc parallel to/sigma magnitude of F0), of 0.132,
0.131, and 0.134, respectively. These inhibitors were designed to be structural
mimics of the tetrahederal transition-state intermediate encountered during
aspartic proteinase catalysis. They are potent inhibitors of penicillopepsin
with Ki values of 1, 22 nM; 2, 2.8 nM; and 3, 1600 nM, respectively [Bartlett,
P. A., Hanson, J. E., & Giannousis, P. P. (1990) J. Org. Chem. 55,
6268-6274]. All three of these phosphorus-based inhibitors bind virtually
identically in the active site of penicillopepsin in a manner that closely
approximates that expected for the transition state [James, M. N. G., Sielecki,
A.R., Hayakawa, K., & Gelb, M. H. (1992) Biochemistry 31, 3872-3886]. The
pro-S oxygen atom of the two phosphonate inhibitors and of the phosphinate group
of the StaP inhibitor make very short contact distances (approximately 2.4 A) to
the carboxyl oxygen atom, O delta 1, of Asp33 on penicillopepsin. We have
interpreted this distance and the stereochemical environment of the carboxyl and
phosphonate groups in terms of a hydrogen bond that most probably has a
symmetric single-well potential energy function. The pro-R oxygen atom is the
recipient of a hydrogen bond from the carboxyl group of Asp213. Thus, we are
able to assign a neutral status to Asp213 and a partially negatively charged
status to Asp33 with reasonable confidence. Similar very short hydrogen bonds
involving the active site glutamic acid residues of thermolysin and
carboxypeptidase A and the pro-R oxygen of bound phosphonate inhibitors have
been reported [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H.,
& Matthews, B. W. (1987) Biochemistry 26, 8542-8553; Kim, H., &
Lipscomb, W. N. (1991) Biochemistry 30, 8171-8180].(ABSTRACT TRUNCATED AT 400
WORDS)
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