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D J Hoover

Publications and source records attributed to D J Hoover.

7 recordsLinked to original sources
PubMed1992

Synthesis and crystallographic analysis of two rhizopuspepsin inhibitor complexes.

The crystal structures of rhizopuspepsin complexed with two oligopeptide inhibitors have been determined. CP-69,799, an azahomostatine dipeptide isostere, had previously been associated with a displacement of the C-terminal subdomain of endothiapepsin [Sali, A., Veerapandian, B., Cooper, J. B., Foundling, S. I., Hoover, D. J., & Blundell, T. L. (1989) EMBO J. 8, 2179-2188]. Here, we report the measurement of two data sets, one from crystals soaked in the inhibitor and the other from protein crystallized in the presence of excess inhibitor. In neither case is there any significant movement of the C-terminal subdomain of the rhizopuspepsin. The data suggest that the energy associated with any conformational change is small and is overcome by the crystal packing forces. The second inhibitor, a hydrated difluorostatone, was examined in a search for transition-state analogs that could cast further light on the mechanism of action [Suguna, K., Padlan, E. A., Smith, C. W., Carlson, W. D., & Davies, D. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7009-7013]. The gem-diol provides a set of contact distances with the enzyme that mimic the interactions with the tetrahedral intermediate of the substrate during catalysis. These data provide support for the suggestion that the polarization of the keto group of the peptide substrate is enhanced by a hydrogen bond from the OD1 of Asp 35 (Suguna et al., 1987).

Amino Acid Sequence↗
PubMed1992

Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.

Amino Acid Sequence↗
PubMed1991

Androgen metabolism by porcine granulosa cells during the process of luteinization in vitro: identification of 19-oic-androstenedione as a major metabolite and possible precursor for the formation of C18 neutral steroids.

The present studies were conducted to define the pathway(s) by which androstenedione is metabolized in porcine granulosa cells (pGC) and determine whether metabolism of this steroid is affected by in vitro luteinization. pGC isolated from large preovulatory follicles were cultured for up to 2 days in the presence of 5 microM unlabeled or [4-14C]-labeled androstenedione. Metabolism of androstenedione was assessed by HPLC, using in-line liquid scintillation detection. Metabolite identification was confirmed by gas chromatography-mass spectrometry of HPLC fractions isolated from medium conditioned by granulosa cells (pGCCM) cultured for 48 h in the presence of unlabeled androstenedione. The metabolites identified were 19-oic-androstenedione (3,17-dioxo-4-androsten-19-oic acid), 19-hydroxytestosterone, 19-hydroxyandrostenedione, 19-nor-testosterone, an estrenolone of as yet unproven stereoisomerism, 5(10)-estrene-3 beta, 17 beta-diol, 17 beta-estradiol, testosterone, and 19-nor-androstenedione. Results indicate that 19-nor-androstenedione is artifactually derived from 19-oic-androstenedione as a result of degradation in storage and during isolation. After metabolite identification, studies of the time course of androstenedione metabolism by pGC during in vitro luteinization were conducted. 17 beta-Estradiol and 19-oic-androstenedione were the predominant metabolites, and accumulation of these steroids was virtually identical. Production of these metabolites was maximal during the first 12 h of culture. The accumulation of 5(10)-estrene-3 beta,17 beta-diol and 19-nor-testosterone was maximal at 48 h of culture, with 5(10)-estrene-3 beta,17 beta-diol consistently accumulating in greater concentrations than 19-nor-testosterone. Aromatase activity of pGC was negligible from 36-48 h of culture, as demonstrated by minimal accumulation of 17 beta-estradiol during this period of culture. The accumulation of 19-oic-androstenedione, 5(10)-estrene-3 beta,17 beta-diol, and 19-nor-testosterone was also negligible during this latter time period, suggesting that their formation is associated with aromatase. From these results, pGC from preovulatory follicles undergoing luteinization in vitro lose the ability to convert androstenedione to estrogens. The formation of 19-oic-androstenedione, shown here for the first time, parallels the formation of 17 beta-estradiol, and this acidic steroid is proposed to be a product of aromatase. As reported in previous studies, pGC do produce C18 neutral steroids from exogenous androstenedione. The production of these steroids requires an active aromatase to produce their immediate precursor, which is here hypothesized to be 19-oic-androstenedione. However, their maximal production does not commence until aromatase activity has declined, and it is hypothesized that their production depends on modifications in steroid metabolism associated with luteinization.

Androgens↗
PubMed1989

High-resolution X-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme.

The conformation of the synthetic renin inhibitor CP-69,799, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 1.8 A resolution and refined to the crystallographic R factor of 16%. CP-69,799 is an oligopeptide transition--state analogue inhibitor that contains a new dipeptide isostere at the P1-P1' position. This dipeptide isostere is a nitrogen analogue of the well-explored hydroxyethylene dipeptide isostere, wherein the tetrahedral P1' C alpha atom has been replaced by trigonal nitrogen. The inhibitor binds in the extended conformation, filling S4 to S3' pockets, with hydroxyl group of the P1 residue positioned symmetrically between the two catalytic aspartates of the enzyme. Interactions between the inhibitor and the enzyme include 12 hydrogen bonds and extensive van der Waals contacts in all the pockets, except for S3'. The crystal structure reveals a bifurcated orientation of the P2 histidine side chain and an interesting relative rotation of the P3 phenyl ring to accommodate the cyclohexyl side chain at P1. The binding of the inhibitor to the enzyme, while producing no large distortions in the enzyme active site cleft, results in small but significant change in the relative orientation of the two endothiapepsin domains. This structural change may represent the action effected by the proteinase as it distorts its substrate towards the transition state for proteolytic cleavage.

Aspartic Acid Endopeptidases↗
PubMedDate not supplied

Ozone deterioration.

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Chemical Phenomena↗