Title

Crystallographic Analysis of Transition State Mimics Bound to Penicillopepsin: Phosphorus-Containing Peptide Analogues

Document Type

Article

Publication Date

6-9-1992

Publication Title

Biochemistry

Department

Chemistry

Abstract

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-(0)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 (= ?||Fo| - |Fc||/?|Fo|), of 0.132, 0.131, and 0.134, respectively. These inhibitors were designed to be structural mimics of the tetrahedral 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, 6268-62741. 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, 3872-38861. The pro-S oxygen atom of the two phosphonate inhibitors and of the phosphinate group of the StaP inhibitor make very short contact distances (~2.4 Å) to the carboxyl oxygen atom, O?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) Bio- chemistry 30, 8 17 1-8 1801. The hydrogen-bonding scheme proposed for the phosphonate inhibitors with the active site of penicillopepsin at pH 4.4 in the crystals can be used to rationalize the pH dependence of the inhibition constant. These data indicate that there are pKa, values near 3.5 and near 5.5 which can be associated with the titration of two protons in the molecular complex. The binding of IvaVVLP(O)FOMe and CbzAALP(O)FOMe to penicillopepsin has allowed, for the first time, experimental characterization of the extensive hydrophobic interactions between the P1’ residue and the S1’ binding site. This structure confirms our proposals for substrate binding to penicillopepsin [James, M. N. G., & Sielecki, A. R. (1985) Biochemistry 24, 3701-37 131. Detailed comparisons of the phosphonate inhibitor binding mode with that of a difluorostatone peptide mimic of the tetrahedral transition state intermediate show that these two inhibitors are shifted by ~0.6 Å relative to one another. In spite of this global shift both inhibitors exhibit equivalently high affinities for the active site of penicillopepsin. Comparison of the binding of IvaVVLP(O)FOMe and CbzAALP(O)FOMe suggests that the origin of the 570-fold difference in binding affinity may be entropic, due to a different number of ordered water molecules displaced in the two cases.

ISSN

0006-2960