Proximity of the substrate binding site and the heme-iron catalytic site in cytochrome P-450scc.

As an approach to "mapping" the active site of the cytochrome P-450 that catalyzes cholesterol side-chain cleavage, designated cytochrome P-450scc, we have synthesized steroid derivatives with the potential to interact with both the substrate binding site and the heme-iron catalytic site of the enzyme. The effects of these substrate analogs were studied with cytochrome P-450scc purified from bovine adrenal cortex. One derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, was found to be a potent inhibitor of pregnenolone formation in a reconstituted enzyme system, and a kinetic analysis of the inhibition showed that binding of the derivative is competitive with respect to cholesterol. The spectral properties of a stable complex formed between the steroidal amine and the purified cytochrome suggest that the 22-amine group coordinates directly to the heme-iron. A model for the structure of this inhibitor-enzyme complex is proposed in which the 5-androstene ring system of the steroid occupies the substrate binding site, and the amine group of the side chain occupies an axial coordination position of the Fe(III) center. This places limits on the distance between these two domains in the enzyme and offers support for proposed mechanisms of cytochrome P-450-catalyzed oxygen-insertion reactions in which an iron-bound oxidant directly attacks the substrate.     

Conformational flexibility at the substrate binding site in the lactose permease of Escherichia coli.

Glu-126 (helix IV) and Arg-144 (helix V) are charge paired and play a critical role in substrate binding in the lactose permease of Escherichia coli. When Glu-126 is replaced with Asp, the permease has relatively high activity, implying that helix V has sufficient flexibility to allow Arg-144 to accommodate the decreased length of the carboxylate-containing side chain of Asp-126. Helices IV and V contain five Gly residues at positions 115, 121, 141, 147, and 150, all of which are conserved in the oligosaccharide/H(+) symport family of the major facilitator superfamily. To test the notion that these residues may contribute to conformational flexibility, each residue was replaced with Ala in either the wild type or the Glu-126 --> Asp mutant. Although the replacements are well tolerated in the wild type, the mutations severely inactivate substrate binding and transport in the Glu-126 --> Asp background, with the exception of Gly-121 --> Ala, which retains significant activity. Strikingly, moreover, in two instances (Gly-150 --> Ala and Gly-141 --> Ala), significant activity is recovered when Ala residues at approximately parallel positions in helix IV (Ala-122 or Ala-127, respectively) are replaced with Gly. In addition to providing further evidence that the major determinants for substrate binding in the permease are at the interface between helices IV and V, the findings indicate that the region is conformationally flexible.     

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

Mitochondrial carriers link biochemical pathways in the cytosol and mitochondrial matrix by transporting substrates across the inner mitochondrial membrane. Substrate recognition is specific for each carrier, but sequence similarities suggest the carriers have similar structures and mechanisms of substrate translocation. By considering conservation of amino acids, distance and chemical constraints, and by modeling family members on the known structure of the ADP/ATP translocase, we have identified a common substrate binding site. It explains substrate selectivity and proton coupling and provides a mechanistic link to carrier opening by substrate-induced perturbation of the salt bridges that seal the pathway to and from the mitochondrial matrix. It enables the substrate specificity of uncharacterized mitochondrial carriers to be predicted.     

The NADPH binding site on beef liver catalase.

Beef liver and human erythrocyte catalases (EC 1.11.1.6) bind NADP tenaciously [Kirkman, H. N. & Gaetani, G. F. (1984) Proc. Natl. Acad. Sci. USA 81, 4343-4348]. The position of NADP on beef liver catalase corresponds to the carboxyl-terminal polypeptide hinge in Penicillium vitale fungal catalase, which connects the common catalase structure to the additional flavodoxin-like domain. In contrast to nearly all other known structures of protein-bound NADP, NAD, and FAD, the NADP molecule of beef liver catalase is folded into a right-handed helix and bound, in part, in the vicinity of the carboxyl end of two alpha-helices. A water molecule (W7) occupies a pseudosubstrate site close to the C4 position of the nicotinamide and is hydrogen bonded to His-304. Although the NADP and heme groups approach each other to within 13.7 A, there is no direct interaction. The function of the NADP remains a mystery.     

NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes.

NMR methods have been adopted to observe directly the characteristics of substrate binding to the galactose-H+ symport protein GalP, in its native environment, the inner membranes of Escherichia coli. Sedimented inner-membrane vesicles containing the GalP protein, overexpressed to levels above 50% of total protein, were analyzed by 13C magic-angle spinning NMR, when in their normal "fluid" state and with incorporated D-[1-13C]glucose. Using conditions of cross-polarization intended to discriminate bound substrate alone, it was possible to detect as little as 250 nmol of substrate added to the membranes containing about 0.5 mumol (approximately 26 mg) of GalP protein. Such high measuring sensitivity was possible from the fluid membranes by virtue of their motional contributions to rapid relaxation recovery of the observed nuclei and due to a high-resolution response that approached the static field inhomogeneity in these experiments. This good spectral resolution showed that the native state of the membranes presents a substrate binding environment with high structural homogeneity. Inhibitors of the GalP protein, cytochalasin B and forskolin, which are specific, and D-galactose, but not L-galactose, prevent or suppress detection of the 13C-labeled glucose substrate, confirming that the observed signal was due to specific interactions with the GalP protein. This specific substrate binding exhibits a preference for the beta-anomer of D-glucose and substrate translocation is determined to be slow, on the 10(-2) s time scale. The work describes a straightforward NMR approach, which achieves high sensitivity, selectivity, and resolution for nuclei associated with complex membrane proteins and which may be combined with other NMR methodologies to yield additional structural information on the binding site for the current transport system without isolating it from its native membrane environment.     

The adsorption of pepsin

Digestive Diseases and Sciences - 1. The most powerful adsorbents of pepsin are aluminum hydroxide gel and charcoal. 2. The adsorbent action of pepsin is uninfluenced by the substrate concentration...     

Elastin sub-fraction as binding site for lipids.

Elastin preparations were isolated from human thoracic aorta, from atherosclerotic and from grossly normal regions. A relatively mild procedure was used to avoid hot alkaline extraction and autoclaving. The elastase digest of the aortic elastin was chromatographed on a Sephadex G-100 column and separated into two fractions: A (larger molecular weight) and B (smaller molecular weight). The ratio of fraction A to total aortic elastin increased with age and the development of the atherosclerosis. Amino acid and sugar analyses showed that fraction A consistently contained more polar amino acids, hexose, hexosamine and L-fucose, and less sialic acid, in comparison with fraction B. Part of the elastin preparation was incubated with human low-density lipoprotein; a considerable amount of lipid, especially cholesterol, was transferred from the lipoprotein to the elastin. Estimation of protein and cholesterol in fractions A and B of the elastase hydrolyzate of incubated elastin showed that most of the cholesterol taken up by elastin had been in fraction A. The increased proportion of fraction A in aortic elastin derived from plaque areas appeared responsible for the marked lipid-binding capacity of plaque elastin.     

Localization of the heparin binding site of follistatin.

To define the heparin-binding site of follistatin, the reduced and S-carboxymethylated recombinant human follistatin containing 288 amino acids was digested by Staphylococcus aureus V8. The digested product was subjected to sulfate cellufine column chromatography and the adsorbed peptide fragments eluted with a stepwise gradient of sodium chloride. The recovered column fractions were further purified by reversed-phase high-performance liquid chromatography (HPLC) and the HPLC peaks subjected to amino-terminal sequence analysis. All of the sulfate cellufine-retarded peptide fragments gave the same N-terminal amino acid sequence, which started at residue-68 of human follistatin, suggested that those fragments starting from residue-68 contain the heparin binding site. The multiple fragments might represent the oxidized, non-glycosylated or glycosylated forms of follistatin(68-113) resulting from the V8 digestion. A synthetic peptide corresponding to the region having the amino acid sequence 72-86 of follistatin was able to bind both heparin and sulfate cellufine, as well as compete with recombinant follistatin for binding to heparin. These findings further define the location of the heparin and heparan sulfate-binding site of follistatin at the basic amino acid-rich region comprising the amino acid sequence Lys75-Lys-Cys-Arg-Met-Asn-Lys-Lys-Asn-Lys-Pro-Arg86.     

The substrate binding domain of DnaK facilitates slow protein refolding

We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states. The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution. The effects of DnaK384-638 on the reactivation of beta-galactosidase and luciferase were examined at low substrate concentration and low temperature, conditions in which the folding is significantly slow (several days) but the reactivation yield is higher than those in ordinary refolding conditions. In the presence of DnaK384-638, the maximum yield of active beta-galactosidase was improved from 45% to 65% after a 48-h refolding reaction. Spectroscopic experiments showed that DnaK384-638 bound to partially structured monomers of beta-galactosidase and consequently suppressed aggregation. DnaK384-638 accelerated the refolding of luciferase to attain equilibrium in 8 h. On the other hand, DnaK386-561, which has no affinity for the substrate, had no chaperone activity for the reactivation of these proteins. These results indicate that the substrate binding of DnaK384-638 facilitates slow protein refolding.     

The dsRNA binding site of human Toll-like receptor 3

Pathogen recognition by Toll-like receptors (TLRs) initiates innate immune responses that are essential for inhibiting pathogen dissemination and for the development of acquired immunity. The TLRs recognize pathogens with their N-terminal ectodomains (ECD), but the molecular basis for this recognition is not known. Recently we reported the x-ray structure for unliganded TLR3-ECD; however, it has proven difficult to obtain a crystal structure of TLR3 with its ligand, dsRNA. We have now located the TLR3 ligand binding site by mutational analysis. More than 50 single-residue mutations have been generated throughout the TLR3-ECD, but only two, H539E and N541A, resulted in the loss of TLR3 activation and ligand binding functions. These mutations locate the dsRNA binding site on the glycan-free, lateral surface of TLR3 toward the C terminus and suggest a model for dsRNA binding and TLR3 activation.     

On the solvent accessibility of substrate binding site of cytochrome P450C-21 in bovine adrenocortical microsomes

The present study offers evidence indicating that acrylamide a polar molecule inhibits substrate-binding to P450C-21 in a competitive manner and quenches tryptophanyl fluorescence in bovine adrenocortical microsomes, similar to that in the purified lipid-free enzyme. Resolution of tryptophanyl fluorescence of the microsomes revealed an acrylamide quenching constant (K2 = 9.9M, is the association constant for the quencher-fluorophore complex) which was similar to the reciprocal of its inhibition constant (1/Kj = Ka = 8.3 +- 0.9M) for substrate-binding. The substrate inhibited the fluorescence quenching by acrylamide which was in accordance with partial competition. In addition the substrate dissociation, acrylamide inhibition and fluorescence quenching constants and tryptophanyl fluorescence maximum (340-342nm) were essentially the same in the microsomes and the purified enzyme. These results suggest that, similar to that in the purified enzyme, a tryptophan in a polar environment in the membrane-bound P450, may serve as a reporter group for the substrate binding site and the site in the membrane-bound enzyme, is accessible to the substrate in aqueous phase.     

Physiological stimulation of pepsin secretion. The role of vagal innervation

The studies were performed on eight double-pouch dogs with one vagally innervated Amdrup pouch (AP) and one denervated Heidenhain pouch (HP), allowing comparison of pepsin secretion from innervated and denervated mucosa at the same time in the same animal. Food stimulation was done with a mixture of liver, heart, and bonemeal in doses of 1.25, 2.5, 5, 10, and 20 g/kg body weight, plus maximal stimulation by repeated meals of 5 g/kg every 15 min up to 10 times. 'Non-active' secretion of pepsin was determined by instillation in the pouches of 0.1 M and 0.005 M HCl dissolved in 0.15 M NaCl. The pepsin secretion in the HP equalled the 'non-active' output for all doses of food. In the innervated pouch there was an immediate, very high pepsin output similar to the response that can be elicited by 2-deoxy-D-glucose and bethanechol chloride. The secretion of pepsin was gradually reduced, but it was, as long as the volume and electrolyte secretion lasted. well above the 'non-active' level. The results show that stimulation of pepsin secretion by food is completely dependent on intact vagal innervation. The volume and acid output were increased as the dose of food was increased, but the concentration of pepsin was stepwise reduced when the volume increased, resulting in approximately the same output of pepsin for all doses of food. The pepsin to acid ratio was statistically higher in juice secreted from innervated than from denervated mucosa for all doses of food, for lower doses of histamine and pentagastrin, and for higher doses of bethanechol chloride.     

MT3/QR2 melatonin binding site does not use melatonin as a substrate or a co-substrate

Quinone reductase 2 (QR2, E.C. 1.10.99.2) is implicated in cell reactive oxygen species production. The catalytic activity of this enzyme is inhibited by 1 microM of melatonin. QR2 was identified as the third melatonin binding site (MT3). It is of major importance to understand the exact roles of melatonin and QR2 in oxidative stress. A fascinating possibility that melatonin could serve as a co-substrate or substrate of QR2 was hypothesized recently. In the current investigation, nuclear magnetic resonance studies of the QR2 catalytic reaction were performed, the results led us to conclude that, whatever the conditions, melatonin is not cleaved off to form N1-acetyl-N2-formyl-5-methoxykynurenine by a catalytically active QR2, very strongly indicating that melatonin is neither a substrate nor a co-substrate of this enzyme. Further studies are needed in order to better understand the relationship between MT3/QR2, melatonin and redox status of the cells, in order to better explain the anti-oxidant activities of melatonin at pharmacological concentrations (>1 microM).     

Eukaryote ribosomes possess a binding site for concanavalin A.

Ribosomes prepared from chicken liver or rabbit reticulocytes bound concanavalin A in a molar ratio of approximately 1:1. This binding is to the large subunit of the eukaryote ribosomes with a dissociation constant of 5 X 10(-7) M (0 degrees). The binding of concanavalin A to Escherichia coli ribosomes was much less. Binding to the RNA or to possible membrane contaminants was ruled out in control experiments. Chicken liver ribosomes were labeled in vivo with 3H-labeled amino acids, purified, and dissociated in sodium dodecyl sulfate. Affinity chromatography of this preparation made it possible to isolate the small proportion of the ribosomal proteins (about 1.5%) containing the concanavalin A binding site. This protein moved as a single band during electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate and showed an apparent molecular weight of 31,000.     

Microenvironment of the binding site in the lac carrier protein.

Studies with a homologous series of (N-dansyl)aminoalkyl-1-thio-beta-D-galactopyranosides containing two to six methylene carbons bridging the galactosyl and dansyl ends of the molecules are described. The compounds were utilized in radioactive and nonradioactive form, and binding of each homologue to membrane vesicles isolated from Escherichia coli ML 308-225 was measured directly by flow dialysis in the presence of D-lactate. The results are compared with the D-lactate-induced fluorescence enhancement observed with each dansylgalactoside and with the ability of N-methylpicolinium perchlorate to quench the fluorescence of the bound homologues. The binding affinity of the lac carrier protein for the probes varies directly with the length of the alkyl linkage, and the same number of binding sites is observed with each homologue. In contrast, however, the increase in fluorescence observed upon binding varies dramatically as the alkyl chain is increased in length, with the fluorescence exhibiting maximal values at two and six methylene carbons and a minimum at four methylene carbons. Furthermore, quenching by N-methylpicolinium perchlorate exhibits an inverse relationship and maximum quenching is observed with the 4 carbon homologue. Possible reasons for this behavior are discussed.