Mechanism of inactivation of 3-oxosteroid delta 5-isomerase by 17 beta-oxiranes

The affinity label (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) inactivates 3-oxosteroid delta 5-isomerase from Pseudomonas testosteroni by formation of a covalent bond between Asp-38 of the enzyme and the steroid. High-performance liquid chromatography (HPLC) analysis of tryptic digests of inactivated enzyme shows that two isomeric steroid-containing peptides are formed in a ratio of 9:1 at pH 7 (TPS1 and TPS2). Hydrolysis of each of these peptides produces a different steroid: TPS1 releases 17 alpha-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 beta-diol (S1) whereas TPS2 yields 17 beta-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 alpha-diol (S2). Inactivation of the enzyme by (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran-18O]-3-ol, followed by mass spectral analysis of the diacetate of the steroid released upon hydrolysis of the enzyme-inhibitor bond, reveals that TPS1 is formed by attack of Asp-38 at the methylene carbon of the oxirane. In contrast, TPS2 is produced by Asp-38 attack at the tertiary carbon. These results imply that inactivation occurs through concurrent SN1 and SN2 reactions of Asp-38 with the protonated inhibitor and that Asp-38 is located on the alpha face of the steroid when it is bound to the active site in the correct manner to react for both the SN1 and SN2 processes.     

Nucleotide sequence of the gene for the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni

The structural gene for the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni has been sequenced by the dideoxy method. The sequence obtained confirms the amino acid (aa) sequence of Benson et al. [J. Biol. Chem. 246 (1971) 7514-7525] at all but 5 aa residues of the 125-aa polypeptide. Amino acid residues 22, 24, 33, and 38, reported to be asparagines by Benson et al., are found to be encoded by aspartic acid codons. Amino acid residue 77, reported to be a glutamine by Benson et al., is encoded by a glutamic acid codon. The identification of aa 38 as aspartic acid, coupled with its presence in the active site, as indicated by previous affinity and photoaffinity-labeling studies and confirmed independently by x-ray crystallographic studies, strengthens the hypothesis that Asp-38 is the aa responsible for the 4 beta to 6 beta proton transfer which is part of the enzymatic reaction.     

The amino acid sequence of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B

We have determined the primary structure of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B. The enzyme is a dimeric protein of two identical subunits, each consisting of a polypeptide chain of 131 residues and a Mr = 14,536. The intact S-carboxymethyl protein was sequenced from the NH2 terminus using standard automated Edman degradation and automated Edman degradation using fluorescamine treatment at known prolines to suppress background. The isomerase was fragmented using CNBr, trypsin, iodosobenzoic acid, and acid cleavage at aspartyl-prolyl peptide bonds. The peptides resulting from each fragmentation were separated by reversed-phase high performance liquid chromatography and sequenced by automated Edman degradation. The full sequence was deduced by the overlapping of the various peptides. A search for homologous proteins was performed. Only the oxosteroid isomerase from Pseudomonas testosteroni, an expected homology, was found to be similar. Comparison of the two proteins shows that the region of strongest homology is the region containing the aspartic acid at which steroidal affinity and photoaffinity reagents have been shown to react in the P. testosteroni isomerase. The P. putida isomerase contains 3 cysteines and 2 tryptophans, whereas the P. testosteroni isomerase lacks these amino acids. The two proteins are not highly conserved.     

Primary structure of human triosephosphate isomerase

Human placental triosephosphate isomerase was isolated by an improved procedure and recovered with the highest specific activity ever reported. Employing this purification procedure, sufficient amounts of the enzyme were obtained for detailed primary structural studies. For sequences analysis, the enzyme was reduced and carboxymethylated and subjected to tryptic and chymotryptic digestions. The peptide mixtures were separated by high-performance liquid chromatography using octyl or alkylphenyl reverse-phase columns and trifluoroacetic acid/acetonitrile gradient elution systems. Sequence analyses of the intact enzyme, tryptic, chymotryptic, and cyanogen bromide peptides were accomplished using high-sensitivity solid-phase sequencing procedures with either 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate or phenylisothiocyanate. The primary structure of human triosephosphate isomerase is constructed from the alignment of the tryptic peptides with the analysis of the overlapping chymotryptic peptides. The enzyme is a dimeric molecule consisting of two identical polypeptide chains with 248 amino acid residues and a calculated subunit molecular mass of 26,750 daltons. A comparison of the amino acid sequences from the human placental enzyme and from other species such as rabbit, chicken, and coelacanth muscles showed relatively high sequence homology, indicating that the evolution of the enzyme is very conservative. The amino acids of the active-site pocket and the subunit-subunit contact sites exhibit few changes.     

Site specificity of histone H4 methylation by wheat germ protein-arginine N-methyltransferase

CNBr treatment of calf thymus [methyl-14C]histone H4, methylated in vitro with S-adenosyl-L-[methyl-14C]methionine by a highly histone-specific wheat germ protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), produced two peptide fragments corresponding to residues 1-83 and 84-102, with the former being radioactive. Two-dimensional peptide mapping of the chymotryptic and tryptic digest of [methyl-14C]histone H4 and analysis of the chymotryptic digest on HPLC have shown that only a single peptide is radiolabeled. In order to define the exact site of methylation (arginine residue), the radioactive peptide from the chymotryptic digest of [methyl-14C]histone H4 was further purified on HPLC by linear and then isocratic elution. The purified chymotryptic peptide was then digested with trypsin and purified on HPLC, and its amino acid composition was determined on HPLC. These results indicate that the peptide corresponding to residues 24-35 of histone H4 is radiolabeled. Since this peptide contains a single arginine residue at position 35, we have concluded that the enzyme is specific not only to the protein substrate but also to the methylation site.     

The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.     

Amino acid sequence of chitinase from Streptomyces erythraeus

The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.     

The primary structure of Lactobacillus casei thymidylate synthetase. II. The complete amino acid sequence of the active site peptide, CNBr 4.

The 102 amino acid residues of CNBr 4, the largest of 5 cyanogen bromide peptides from the Lactobacillus casei thymidylate synthetase were completely sequenced by means of limited tryptic, tryptic, chymotryptic, and staphylococcal protease peptides. CNBr 4 contains both of the cysteines in an enzyme subunit, with the 5-fluorodeoxyuridylate-reactive cysteine at residue 198 and the other at residue 244.     

Identification of the apparently essential lysine residues in phospholipase C (Bacillus cereus).

Phospholipase C (Bacillus cereus) contains two apparently essential and very reactive lysine residues that may be labelled selectively by pyridoxal 5'-phosphate [Aurebekk & Little (1977) Biochem, J. 161, 159--165]. One of these lysine residues was found in the 25-amino acid N-terminal fragment liberated by CNBr digestion of the pyridoxal-labelled enzyme and identified as lysine-6. Two of the labelled peptides isolated from the chymotryptic digest of pyridoxal-labelled enzyme contained proline, suggesting that the other labelled lysine residue is situated in the same region of the primary structure as the single proline residue of the enzyme.     

Binding-site specificity of the radiolytically induced crosslinking of phenylalanine to glucagon

The gamma-radiation-induced crosslinking of phenylalanine to glucagon, mediated by OH ., has been shown to involve a limited number of binding sites on the glucagon molecule. Glucagon-phenylalanine adducts were partially separated from other radiolysis products with Sephadex gel filtration; further isolation of adducts was achieved with reverse-phase high-performance liquid chromatography (HPLC). Amino acid analysis of the isolated adducts indicates that the aromatic residues (phenylalanine and tyrosine), basic residues (histidine and lysine), and sulfur-containing residue (methionine) of glucagon are predominantly involved in crosslinking; these are essentially the same residues implicated in glucagon-glucagon crosslinking. Acid hydrolysates and chymotryptic digests of glucagon-phenylalanine adducts were examined with HPLC. The number of amino acid-phenylalanine adducts and also chymotryptic peptides observed was much greater than would have been expected based on the amino acid analysis. This observation is best accounted for by the involvement in crosslinking of radicals formed on the glucagon with more than one possible phenylalanine-derived free radical.     

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.     

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.     

Isopentenyl-diphosphate isomerase: irreversible inhibition by 3-methyl-3,4-epoxybutyl diphosphate.

Isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl diphosphate (IPP) to its allylic isomer, dimethylallyl diphosphate (DMAPP). Incubation of yeast IPP isomerase with 3-methyl-3,4-epoxybutyl diphosphate (EIPP) resulted in a time-dependent first-order loss of activity characteristic of an active-site-directed irreversible process, where k2 = 0.63 +/- 0.10 min-1 and KI = 0.37 +/- 0.11 microM. A 1:1 covalent E-I complex was formed upon incubation with [1-14C]EIPP. The inhibited enzyme was treated with trypsin to give two radioactive fragments, which were purified by reversed-phase HPLC on a C18 column. The modified amino acid in each fragment was identified as C139 by sequencing the radiolabeled peptides. Incubation of IPP isomerase with [2,4,5-13C3]EIPP gave a 13C-labeled E-I complex. A 1H-13C heteronuclear multiquantum correlation spectrum had strong cross-peaks at 1.2/28 and 2.9/48 ppm, which we assigned to the labeled methyl group and C(4) methylene, respectively, of the inhibitor. In addition, a weak signal at 2.17/42 ppm may be from the C(2) methylene. Comparison of these chemical shifts with those of a synthetic adduct isolated from treatment of EIPP with cysteine indicates C139 attacks C(4) of EIPP to generate a thioether linkage between the enzyme and the inhibitor.     

Glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase (EC 5.3.1.9) is a dimeric enzyme of molecular mass 132000 which catalyses the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. The crystal structure of the enzyme from pig muscle has been determined at a nominal resolution of 2.6 A. The structure is of the alpha/beta type. Each subunit consists of two domains and the active site is in both the domain interface and the subunit interface (P.J. Shaw & H. Muirhead (1976), FEBS Lett. 65, 50-55). Each subunit contains 13 methionine residues so that cyanogen bromide cleavage will produce 14 fragments, most of which have been identified and at least partly purified. Sequence information is given for about one-third of the molecule from 5 cyanogen bromide fragments. One of the sequences includes a modified lysine residue. Modification of this residue leads to a parallel loss of enzymatic activity. A tentative fit of two of the peptides to the electron density map has been made. It seems possible that glucose-6-phosphate isomerase, triose phosphate isomerase and pyruvate kinase all contain a histidine and a glutamate residue at the active site.     

Chemical modification of chalcone isomerase by diethyl pyrocarbonate: histidine residues are not essential for catalysis

Chalcone isomerase form soybean is inactivated by treatment with diethyl pyrocarbonate (DEP). The competitive inhibitor 4',4-dihydroxychalcone provides kinetic protection against inactivation by DEP with a binding constant at the site of protection in agreement with its binding constant at the active site. Very high concentrations of the competitive inhibitors 4',4-dihydroxychalcone or morin hydrate offer a 10- to 40-fold maximal protection, suggesting a second slower mechanism for inactivation which cannot be prevented by blockage of the active site. Blockage of the only cysteine residue in chalcone isomerase with p-mercuribenzoate does not affect the rate constant for DEP-dependent inactivation and indicates that the modification of the cysteine residue is not responsible for the activity loss observed in the presence of DEP. Treatment of inactivated enzyme with hydroxylamine does not restore catalytic activity, indicating that the modification of histidine or tyrosine residues is not responsible for the activity loss. All five histidines of chalcone isomerase are modified by DEP at pH 5.7 and ionic strength 1.0 M. The rate constant for the modification of the histidine residues of chalcone isomerase is close to that for the reaction of N-acetyl histidine with DEP, indicating that the histidine residues are quite accessible to the modifying reagent. The rate of histidine modification is the same in native enzyme, in urea-denatured enzyme, and in the presence of a competitive inhibitor. In the presence of the competitive inhibitor morin hydrate, all of the histidine residues of chalcone isomerase can be modified without significant loss in catalytic activity. These results demonstrate that the histidine residues of chalcone isomerase are not essential for catalysis and therefore cannot function as nucleophilic catalysts as previously proposed.     

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors.

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.     

A reinvestigation of residues 64–68 and 175 in papain. Evidence that residues 64 and 175 are asparagine

The tryptophan-containing peptides were isolated from the chymotryptic digest of S-carboxymethylated papain. Residue 175, which is strongly hydrogen-bonded to the active-site histidine residue in the tertiary structure of papain, is asparagine, confirming the work of Kimmel, Rogers & Smith (1965). Its function is probably to maintain the orientation and tautomeric state of the imidazole ring of histidine-159. The amino acid sequence predicted from the electron-density map of papain for residues 64-68 was confirmed, but residue 64 is asparagine, not aspartic acid. This residue, which is about 10 A from the thiol group of the active-site cysteine-25, cannot therefore be a site of electrostatic attraction for substrates of basic amino acids.     

Bacillus cereus 569/H penicillinase serine-44 acylation by diazotized 6-aminopenicillanic acid

Penicillinase from Bacillus cereus 569/H was purified to homogeneity. Its active site was probed by use of an affinity label generated in situ by the diazotization of 6-aminopenicillanic acid, a catalytically poor substrate for this enzyme. The loss of activity arising during the inactivation is dependent upon pH and the penicillin:sodium nitrite ratio used. Optimal inactivation was obtained at pH 4.7 and reactivation could be prevented if subsequent purification and manipulations were performed at low pH. Inactivation by diazotized 6-aminopenicillanic acid was characterized further by tryptic and chymotryptic digestion of the inactivated enzyme and peptide mapping of the resulting digests. Amino acid analysis of the chymotryptic labeled peptide yielded a composition which corresponds to residues 41-46 (Ala-Phe-Ala-Ser-Thr-Tyr) in the published partial sequence of the enzyme (Thatcher, D. (1975) Biochem. J. 147, 313-326). Further digestion of this chymotryptic peptide with carboxypeptidase A reveals that serine-44 is modified in this affinity labeling procedure. Mass spectral analysis of the modified serine residue and alkali-released label, and comparison with spectra of model compounds indicates that the inactivation occurs with rearrangement of the beta-lactamthiazolidine structure to a dihydrothiazine.     

Stereochemistry of the concerted enolization catalyzed by delta 5-3-ketosteroid isomerase

The reaction catalyzed by delta 5-3-ketosteroid isomerase has been shown to occur via the concerted enolization of the delta 5-3-ketosteroid substrate to form a dienolic intermediate, brought about by Tyr-14, which hydrogen bonds to and protonates the 3-keto group, and Asp-38, which removes and axial (beta) proton from C-4 of the substrate, in the same rate-limiting step [Xue, L., Talalay, P., & Mildvan, A.S. (1990) Biochemistry 29, 7491-7500; Kuliopulos, A., Mildvan, A.S., Shortle, D., & Talalay, P. (1989) Biochemistry 26, 3927-3937]. Since the axial C-4 proton is removed by Asp-38 from above the substrate, a determination of the complete stereochemistry of this rapid, concerted enolization requires information on the direction of approach of Tyr-14 to the enzyme-bound steroid. The double mutant enzyme, Y55F + Y88F, which retains Tyr-14 as the sole Tyr residue, was prepared and showed only a 4.5-fold decrease in kcat (12,000 s-1) and a 3.6-fold decrease in KM (94 microM) for delta 5-androstene-3, 17,dione, in comparison with the wild-type enzyme. Deuteration of the aromatic rings of the 10 Phe residues further facilitated the assignment of the aromatic proton resonances of Tyr-14 in the 600-MHz TOCSY spectrum at 6.66 +/- 0.01 ppm (3,5H) and at 6.82 +/- 0.01 ppm (2,6H). Variation of the pH from 4.9 to 10.9 did not alter these shifts, indicating that the pKa of Tyr-14 exceeds 10.9. Resonances assigned to the three His residues titrated with pKa values very similar to those found with the wild-type enzyme. The binding of 19-nortestosterone, a product analogue and substrate of the reverse isomerase reaction, induced downfield shifts of -0.12 and -0.06 ppm of the 3,5-and 2,6-proton resonances of Tyr-14, respectively, possibly due to deshielding by the 3-keto group of the steroid, but also induced +0.29 to -0.41 ppm changes in the chemical shifts of 8 of the 10 Phe residues and smaller changes in 10 of the 12 ring-shifted methyl resonances, indicating a steroid-induced conformation change in the enzyme. NOESY spectra in H2O revealed strong negative Overhauser effects from the 3,5-proton resonance of Tyr-14 to the overlapping 2 alpha-, 2 beta-, or 6 beta-proton resonances of the bound steroid but no NOE's to the 4- or 6 alpha-protons of the steroid.(ABSTRACT TRUNCATED AT 400 WORDS)