Synthesis and biological activity of new conformationally restricted analogues of pepstatin.

A new statine derivative, 3-hydroxy-4-amino-5-mercaptopentanoic acid; cysteinylstatine (CySta), was synthesized and used to prepare a series of conformationally restricted analogues of pepstatin (Iva-Val-Val-Sta-Ala-Sta) in which the conformational constraint was introduced via a bis-sulfide connecting the appropriately substituted residues in the P1 and the P3 inhibitor side chains. The precursor peptide, Iva-Cys-Val-CySta-Ala-Iaa, was synthesized and alkylated with a series of dibromoalkanes and alkenes to produce the cyclic structures. This strategy permitted the carbon atom spacing between the P1 and the P3 inhibitor side chains to be systematically varied so as to produce inhibitors with 15-, 16-, and 17-membered ring systems. Additional non-cyclic analogues were synthesized as controls by alkylating the bisthiol intermediates with methyl iodide. The inhibitory potency of the analogues were determined against porcine pepsin and penicillopepsin by using standard enzyme kinetic assays. The cyclic inhibitor were found to be potent inhibitors of both aspartic proteases; inhibitor that contained a trans-2-butene link between the two sulfur atoms was found to be the most potent inhibitor with a Ki less than 1 nM against pepsin and 3.94 nM against penicillopepsin. This series of compounds illustrates a new type of conformational restriction that can be used to probe for the bioactive conformation of peptides.     

Biological activity of aspartic proteinase inhibitors related to pepstatin

We have synthesized eight tripeptide analogs of pepstatin in which both the side-chain and stereochemistry of the novel amino acid statine have been altered. They have been compared to pepstatin for inhibition of pepsin and cathepsin D activity, inhibition of autolysis at pH 4, and inhibition of protein degradation in cultured cells. Effective inhibition of aspartic proteinase activity appears to require the novel amino acid to have a bulky hydrophobic side-chain and the S-configuration at both chiral centers. However, the Cbz-Val-Val-(3S4S)-statine peptide was more effective than pepstatin in cultured cells, and inhibition was also achieved, and in some cases enhanced relative to pepstatin, by its stereoisomers and by tripeptides containing valyl and alanyl analogs of statine.     

Investigation of the synthetic route to pepstatin analogues by SPPS using O‐protected and O‐unprotected statine as building blocks

The synthetic route to pepstatin derivatives by a solid phase peptide synthesis using either O‐protected or O‐unprotected statine as a building block has been investigated. Statine was prepared according to a modified literature procedure, whereas protection of its 3‐hydroxyl moiety using tert‐butyldimethylsilylchloride (TBSCl) provided the novel O‐TBS‐protected statine building block. The O‐tert‐butyldimethylsilyl (TBS)‐protected statine approach provides an improved synthetic strategy for the preparation of statine‐containing peptides as demonstrated by the synthesis of the pepstatin analogue iva‐Val‐Leu‐Sta‐Ala‐Sta. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular model for the binary complex of uropepsin and pepstatin

The three-dimensional structure of human uropepsin complexed with pepstatin has been modelled using human pepsin as a template. Uropepsin is an aspartic proteinase from the urine, produced in the form of pepsinogen A in the gastric mucosa. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo twofold axis. A structural comparison between binary complexes of pepsin:pepstatin and uropepsin:pepstatin is discussed.     

Fluoro ketone inhibitors of hydrolytic enzymes

The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.     

New renin inhibitors homologous with pepstatin.

Four homologues of pepstatin, the potent but poorly soluble inhibitor of aspartic proteinases, were synthesized by coupling to the C-terminus of the natural pentapeptide the following amino acid residues: L-arginine methyl ester, L-aspartic acid, L-glutamic acid and the dipeptide L-aspartyl-L-arginine. The peptide-coupling reagent we used, benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate, allowed us to obtain readily pure pepstatin homologues with high yields (60-83%). Pepstatylarginine methyl ester and pepstatylglutamic acid were about one order of magnitude more water-soluble than pepstatin. The four homologues and pepstatin were tested in vitro as inhibitors for highly purified pig and human renins acting on the N-acetyltetradecapeptide substrate. The 50% inhibitory concentrations (IC50) of the homologues were ranged from 0.01 to 1 microM against porcine renin at pH 6.0 (pepstatin IC50 approximately 0.32 microM) and from 5.8 to 41 microM against human renin at pH 6.5 (pepstatin IC 50 approximately 17 microM). By three different graphical methods we showed that pepstatin and the four homologues behaved as competitive inhibitors for porcine renin. The most potent inhibitors were pepstatylaspartic acid and pepstatylglutamic acid, with inhibitory constants respectively 2- and 10-fold smaller than that of pepstatin. By coupling glutamic acid to pepstatin, the ratio solubility/Ki was increased by two orders of magnitude.     

Synthesis of A 3-oxo-4(S)-amino acid analog of pepstatin. A new inhibitor of carboxyl (acid) proteases

A ketone analog of pepstatin, in which the 3S hydroxyl group is oxidized to a ketone group, has been synthesized and shown to be a potent inhibitor of pepsin. Kinetics of inhibition of pepsin provide evidence that the ketone pepstatin analog binds to pepsin differently than pepstatin. The relationship of these complexes to crystal complexes of pepstatin-carboxyl proteases is discussed.     

Crystal structure of human pepsin and its complex with pepstatin.

The three-dimensional crystal structure of human pepsin and that of its complex with pepstatin have been solved by X-ray crystallographic methods. The native pepsin structure has been refined with data collected to 2.2 A resolution to an R-factor of 19.7%. The pepsin:pepstatin structure has been refined with data to 2.0 A resolution to an R-factor of 18.5%. The hydrogen bonding interactions and the conformation adopted by pepstatin are very similar to those found in complexes of pepstatin with other aspartic proteinases. The enzyme undergoes a conformational change upon inhibitor binding to enclose the inhibitor more tightly. The analysis of the binding sites indicates that they form an extended tube without distinct binding pockets. By comparing the residues on the binding surface with those of the other human aspartic proteinases, it has been possible to rationalize some of the experimental data concerning the different specificities. At the S1 site, valine at position 120 in renin instead of isoleucine, as in the other enzymes, allows for binding of larger hydrophobic residues. The possibility of multiple conformations for the P2 residue makes the analysis of the S2 site difficult. However, it is possible to see that the specific interactions that renin makes with histidine at P2 would not be possible in the case of the other enzymes. At the S3 site, the smaller volume that is accessible in pepsin compared to the other enzymes is consistent with its preference for smaller residues at the P3 position.     

Novel inhibition of porcine pepsin by a substituted piperidine. Preference for one of the enzyme conformers

Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3R,4R)-3-(4-bromobenzyloxy)-4-[4-(2-naphthalen-1-yl-2-oxo-ethoxy)phenyl]piperidine) has been studied using steady-state kinetic and pre-equilibrium binding methods. Data were compared with pepstatin A, a well known competitive inhibitor of pepsin. Steady-state analysis reveals that the substituted piperidine likewise behaves as a competitive inhibitor. Pre-equilibrium binding studies indicate that the substituted piperidine can displace a fluorescently labeled statine inhibitor from the enzyme active site. Simulation of the stopped-flow fluorescence transients provided estimates of the K(d) values of 1.4 +/- 0.2 microm and 39 +/- 2 nm for the piperidine and the fluorescently labeled statine, respectively. The effects of combinations of these two inhibitors resulted in a series of parallel lines when plotted by the method of Yonetani and Theorell (Yonetani, T., and Theorell, H. (1964) Arch. Biochem. Biophys. 106, 234-251), suggesting that the two inhibitors bind in a mutually exclusive fashion to pepsin. Fitting of the entire data set to the appropriate equation yielded an alpha factor of 8 +/- 1. The magnitude of this factor ( infinity > alpha > 1) can be explained by a conformational distinction between the enzyme species that bind each inhibitor. The effects of pH on the inhibition constants for pepstatin A and the substituted piperidine also suggest that the inhibitors bind to distinct conformational forms of the enzyme. No inhibition by the piperidine was observed at acidic pH, while pepstatin A inhibition is maximal at low pH values. Inhibition by the piperidine was maximal when a group with pK 4.8 +/- 0.2 was deprotonated and another group with pK 5.9 +/- 0.2 was protonated. Most likely these two groups are the catalytic aspartates with perturbed ionization properties as a result of a significant and unique conformational change. Taken together, these data suggest that the enzyme can readily interconvert between two conformers, one capable of binding substrate and pepstatin A and the other capable of binding the substituted piperidine.     

A novel cell penetrating aspartic protease inhibitor blocks processing and presentation of tetanus toxoid more efficiently than pepstatin A

Selective inhibition of enzymes involved in antigen processing such as cathepsin E and cathepsin D is a valuable tool for investigating the roles of these enzymes in the processing pathway. However, the aspartic protease inhibitors, including the highly potent pepstatin A (PepA), are inefficiently transported across the cell membrane and thus have limited access to antigen processing compartments. Previously described mannose-pepstatin conjugates were efficiently taken up by the cells via receptor mediated uptake. However, cells without mannose receptors are unable to take up these conjugates efficiently. The aim of the present study was to synthesize new cell-permeable aspartic protease inhibitors by conjugating pepstatin A with well-known cell penetrating peptides (CPPs). To achieve this, the most commonly used CPPs namely pAntp_(43-58) (penetratin), Tat_(49-60), and 9-mer of l-arginine (R9), were synthesized and coupled to pepstatin. The enzyme inhibitory properties of these bioconjugates and their cellular uptake into MCF7 (human breast cancer cell line), Boleths (EBV-transformed B-cell line) and dendritic cells (DC) were the focus of our study. We found that the bioconjugate PepA-penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor tested, and was more efficient than unconjugated PepA. Additionally, we found that PepA-P efficiently inhibited the tetanus toxoid C-fragment processing in peripheral blood mononuclear cells (PBMC), primary DC and in primary B cells. Therefore, PepA-P can be used in studying the role of intracellular aspartic proteases in the MHC class II antigen processing pathway. Moreover, inhibition of tetanus toxoid C-fragment processing by PepA-P clearly implicates the role of aspartic proteinases in antigen processing.     

Two-step binding mechanism for HIV protease inhibitors.

Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-Phe psi-[CH(OH)CH2N]Pro-O-t-Bu (R diastereomer = 1R; S diastereomer = 1S) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu psi(CH2NH)Leu-Gln-Arg-NH2 (2), (carbobenzyloxy)-Phe psi[CH(OH)CH2N]Pro-O-t-Bu (3), and pepstatin were determined by trapping free enzyme with 1R as 2, 3, and pepstatin dissociated from the respective enzyme.inhibitor complex. Association rate constants of 1R, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO2)-Gln-Arg-NH2 (4). The kinetic data for binding of 1S to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values.     

Inhibition of XMRV and HIV-1 proteases by pepstatin A and acetyl-pepstatin

The kinetic properties of two classical inhibitors of aspartic proteases (PRs), pepstatin A and acetyl-pepstatin, were compared in their interactions with HIV-1 and xenotropic murine leukemia virus related virus (XMRV) PRs. Both compounds are substantially weaker inhibitors of XMRV PR than of HIV-1 PR. Previous kinetic and structural studies characterized HIV-1 PR-acetyl-pepstatin and XMRV PR-pepstatin A complexes and suggested dramatically different binding modes. Interaction energies were calculated for the possible binding modes and suggested a strong preference for the one-inhibitor binding mode for HIV-1 PR-acetyl-pepstatin and the two-inhibitor binding mode for XMRV PR-pepstatin A interactions. Comparison of the molecular models suggested that in the case of XMRV PR the relatively unfavorable interactions at S3' and the favorable interactions at S4 and S4' sites with the statine residues may shift the ground state binding towards the two-inhibitor binding mode, whereas the single molecule ground state binding of statines to the HIV-1 PR appear to be more favorable. The preferred single molecular binding to HIV-1 PR allows the formation of the transition state complex, represented by substantially better binding constants. Intriguingly, the crystal structure of the complex of acetyl-pepstatin with XMRV PR has shown a mixed type of binding: the unusual binding mode of two molecules of the inhibitor to the enzyme, in a mode very similar to the previously determined complex with pepstatin A, together with the classical binding mode found for HIV-1 PR. The structure is thus in good agreement with the very similar interaction energies calculated for the two types of binding. Database The final coordinates of the crystal structure of XMRV protease complexed with acetyl-pepstatin are available in the Protein Data Bank under the accession number 4EXH Structured digital abstract ? HIV-1 PR?and?HIV-1 PR?bind?by?biochemical?(View interaction) ? XMRV PR?cleaves?MLV Gag?by?enzymatic study?(View interaction) ? XMRV PR?and?XMRV PR?bind?by?biochemical?(View interaction) ? XMRV PR?and?XMRV PR?bind?by?x-ray crystallography?(View interaction).     

Three-dimensional structure of the complex of the Rhizopus chinensis carboxyl proteinase and pepstatin at 2.5-A resolution

An X-ray diffraction analysis has been carried out at 2.5-A resolution of the three-dimensional structure of the Rhizopus chinensis carboxyl proteinase complexed with pepstatin. The resulting model of the complex supports the hypothesis [Marciniszyn, J., Hartsuck, J.A., & Tang, J. (1976) J. Biol. Chem. 251, 7088-7094] that statine (3-hydroxy-4-amino-6-methylheptanoic acid) approaches an analogue of the transition state for catalysis. The way in which pepstatin binds to the enzyme can be extended to provide a model of substrate binding and a model of the transition-state complex. This in turn has led to a proposed mechanism of action based on general acid-base catalysis with no covalent intermediates. These predictions are in general agreement with kinetic studies using several carboxyl proteinases, which together with their sequence homology and their common three-dimensional structures suggest that this mechanism can be extrapolated to all carboxyl proteinases.     

Crystal structure of aspartic proteinase from Irpex lacteus in complex with inhibitor pepstatin

The crystal structure of Irpex lacteus aspartic proteinase (ILAP) in complex with pepstatin (a six amino acid residue peptide-like inhibitor) was determined at 1.3A resolution. ILAP is a pepsin-like enzyme, widely distributed in nature, with high milk-clotting activity relative to proteolytic activity. The overall structure was in good topological agreement with pepsin and other aspartic proteases. The structure and interaction pattern around the catalytic site were conserved, in agreement with the other aspartic proteinase/inhibitor complex structures reported previously. The high-resolution data also supported the transition state model, as proposed previously for the catalytic mechanism of aspartic proteinase. Unlike the other aspartic proteinases, ILAP was found to require hydrophobic residues either in the P(1) or P(1') site, and also in the P(4) and/or P(3) site(s) for secondary interactions. The inhibitor complex structure also revealed the substrate binding mechanism of ILAP at the P(3) and P(4) site of the substrate, where the inserted loop built up the unique hydrophobic pocket at the P(4) site.     

Design, synthesis, and biological evaluation of 1,3-diarylprop-2-en-1-ones : a novel class of cyclooxygenase-2 inhibitors

A group of regioisomeric (E)-1,3-diarylprop-2-en-1-one derivatives possessing a COX-2 SO2Me pharmacophore at the para position of the C-1 or C-3 phenyl ring, in conjunction with a C-3 or C-1 phenyl (4-H) or substituted-phenyl ring (4-F, 4-OMe and 4-Me), were designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. These target (E)-1,3-diarylprop-2-en-1-ones were synthesized via a Claisen-Schmidt condensation reaction. In vitro COX-1/COX-2 isozyme inhibition structure-activity studies identified (E)-1-(4-methanesulfonylphenyl)-3-(4-methylphenyl)prop-2-en-1-one (9f) as a potent COX-2 inhibitor (IC50=0.3 microM) with a high COX-2 selectivity index (SI=106) comparable to that of the reference drug rofecoxib (COX-2 IC50=0.5 microM; COX-2 SI>200). A molecular modeling study where 9f was docked in the binding site of COX-2 showed that the para-SO2Me substituent on the C-1 phenyl ring is oriented in the vicinity of the secondary COX-2 binding site near Val523. The structure-activity data acquired indicate that the propenone moiety constitutes a suitable scaffold to design novel acyclic 1,3-diarylprop-2-en-1-ones with selective COX-2 inhibitory activity.     

Crystallographic analysis of transition state mimics bound to penicillopepsin: difluorostatine- and difluorostatone-containing peptides.

Difluorostatine- and difluorostatone-containing peptides have been evaluated as potent inhibitors of penicillopepsin, a member of the aspartic proteinase family of enzymes. Isovaleryl-Val-Val-StaF2NHCH3 [StaF2 = (S)-4-amino-2,2-difluoro-(R)-3-hydroxy-6-methylheptanoic acid] and isovaleryl-Val-Val-StoF2NHCH3 [StoF2 = (S)-4-amino-2,2-difluoro-3-oxo-6-methylheptanoic acid] have measured Ki's of 10 x 10(-9) and 1 x 10(-9) M, respectively, with this fungal proteinase. The StoF2-containing peptide binds 32-fold more tightly to the enzyme than the analogous peptide containing the non-fluorinated statine ethyl ester. Each compound was cocrystallized with penicillopepsin, intensity data were collected to 1.8-A resolution, and the atomic coordinates were refined to an R factor [formula: see text] of 0.131 for both complexes. The inhibitors bind in the active site of penicillopepsin in much the same fashion as do other statine-containing inhibitors of penicillopepsin analyzed earlier [James, M. N. G., Sielecki, A. Salituro, F., Rich, D. H., & Hofmann, T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6137-6141; James, M.N.G., Sielecki, A., & Hofmann, T. (1985) in Aspartic Proteinases and their Inhibitors (Kosta, V., Ed.) pp 163-177, Walter deGruyter, Berlin]. The (R)-3-hydroxyl group in StaF2 binds between the active site carboxyl groups of Asp33 and Asp213, making hydrogen-bonding contacts to each one. The ketone functional group of the StoF2 inhibitor is bound as a hydrated species, with the gem-diol situated between the two aspartic acid carboxyl groups in a manner similar to that predicted for the tetrahedral intermediate expected during the catalytic hydrolysis of a peptide bond [James, M. N. G., & Sielecki, A. (1985) Biochemistry 24, 3701-3713]. One hydrogen-bonding interaction from the "outer" hydroxyl group is made to O delta 1 of Asp33, and the "inner" hydroxyl group forms two hydrogen-bonding contacts, one to each of the carboxyl groups of Asp33 (O delta 2) and Asp213 (O delta 2). The only structural difference between the StaF2 and StoF2 inhibitors that accounts for the factor of 10 in their Ki's is the additional (R)-3-OH group on the tetrahedral sp3 carbon atom of the hydrated StoF2 inhibitor. The intermolecular interactions involving the fluorine atoms of each inhibitor are normal van der Waals contacts to one of the carboxyl oxygen atoms of Asp213 (F2-O delta 2 Asp213, 2.9 A). The observed stereochemistry of the bound StoF2 group in the active site of penicillopepsin has stimulated our reappraisal of the catalytic pathway for the aspartic proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Miraziridine A: natures blueprint towards protease class-spanning inhibitors

The natural product miraziridine A isolated from the marine sponge Theonella aff. mirabilis unifies within one molecule three structurally privileged elements: (i) (2R,3R)-aziridine-2,3-dicarboxylic acid, (ii) (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (statine), and (iii) (E)-(S)-4-amino-7-guanidino-hept-2-enoic acid (vinylogous arginine). The alignment of them realized in the tetrapetide allows for a simultaneous inhibition of the proteolytic activity of trypsin-like serine proteases, papain-like cysteine proteases, and pepsin-like aspartyl proteases. Therefore, this unique compound represents a blueprint for the design of protease class-spanning inhibitors.     

1H, 13C,and 15N backbone resonance assignments of the porcine pepsin and porcine pepsin complexed with pepstatin

Pepsin is formed as the zymogen, pepsinogen, which includes an additional 44 residue prosegment (PS) on the N-terminus. Upon acidification (pH <3) the PS is removed, yielding active pepsin. The PS is critical to such processes as the initiation of correct folding and protein stability. In the present study, the NMR assignments of the 34.6 kDa native porcine pepsin and porcine pepsin complexed with pepstatin are reported in order to obtain structural information regarding PS-catalyzed protein folding. Such information would contribute to a better understanding of the nature of folding/unfolding energy barrier of pepsin and other aspartic proteases.     

The selectivity of statine-based inhibitors against various human aspartic proteinases.

The interactions of five human enzymes (renin, pepsin, gastricsin, cathepsin D and cathepsin E) and the aspartic proteinase from Endothia parasitica with several series of synthetic inhibitors were examined. All of the inhibitors contained the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues in the P1-P1' positions. The residues occupying the peripheral sub-sites (P4 to P3') were varied systematically and inhibitory constants were determined for the interactions with each of the proteinases. Inhibitors were elucidated that specifically inhibited human renin and did not affect any of the other human enzymes or the fungal proteinase. With suitable selection of residues to occupy individual sub-sites, effective inhibitors of specific human aspartic proteinases may now be designed.     

Amino-steroids as inhibitors and probes of the active site of cytochrome P-450scc. Effects on the enzyme from different sources

A series of analogues of cholesterol, each having a primary amine attached to a shortened side chain, were tested for their effects on cytochrome P-450scc from several different sources. Reconstituted enzyme systems using disrupted mitochondria from bovine adrenal and placenta, adult human adrenal and placenta, neonatal human adrenal, and rat adrenal and testis were used to assay for inhibitory effects on the side chain cleavage of cholesterol to pregnenolone. Two of the derivatives tested, 22-amino-23,24-bisnor-5-cholen-3 beta-ol and 23-amino-24-nor-5-cholen-3 beta-ol, were found to be potent inhibitors of this reaction; the derivatives in which the amine was attached closer to or further from the steroid ring, (20 R and S)-20-amino-5-pregnen-3 beta-ol and 24-amino-5-cholen-3 beta-ol, were much weaker inhibitors. In addition, spectral studies with rat adrenal mitochondria and a soluble preparation of human placental cytochrome P-450scc showed that binding of the 22-amine derivative to the enzyme produces difference spectra characteristic of nitrogen bonding to the heme; this indicates that the heme is positioned close to C-22 in the steroid-enzyme complex. These findings on the relative effectiveness of the amino-steroid inhibitors and the type of complex formed are similar to results obtained with purified bovine adrenocortical cytochrome P-450scc. This establishes that the proximity of the substrate binding site and the heme-iron catalytic site is a feature common to the enzyme from several sources and is therefore likely to be a necessary property of the active site structure.