Adrenodoxin reductase-adrenodexin complex.

Adrenodoxin reductase and adrenodoxin have been shown (Chu, J.-W., and Kimura, T. (1973) J. Biol. Chem. 248, 5183-5187) to form a low dissociation constant, 1:1 complex when both proteins are in the oxidized form. We have found that when adrenodoxin: adrenodoxin reductase ratios are varied by increasing the adrenodoxin concentration, with adrenodoxin reductase held constant, an increasing rate of cytochrome c reduction, with NADPH as reductant, is seen up to a ratio of 1:1, indicating that cytochrome c reduction occurs via the protein-protein complex. Spectra observed during titration of this protein-protein complex with NADH were resolved into components by the linear programming method, using a computer program written in Fortran IV. Analysis of the data has shown that the flavoprotein is reduced prior to the iron sulfur protein, and that the midpoint oxidation-reduction potentials (pH 7.5) of the two proteins are -295 and -331 mV, respectively, when both are present in the complex. Complex formation does not alter the potential of adrenodoxin reductase, but changes that of adrenodoxin by -40 mV. Equilibrium constants derived from potential measurements show that the strength of the protein-protein interaction in the complex is unaltered by reduction of adrenodoxin reductase, but is decreased by about 1 kcal due to reduction of adrenodoxin. The low dissociation constants for both oxidized reduced forms of the adrenodoxin reductase-adrenodoxin complex indicate that the complex must remain associated throughout its catalytic cycle. Titration of the adrenodoxin reductase-adrenodoxin complex with the physiologic reductant, NADPH, was followed by EPR and visible spectra, and yielded an order of reduction of the components identical with that seen when NADH was used as reductant. Reduction of the protein-protein complex with NADPH yielded a ternary complex between NADP+, flavoprotein, and iron sulfur protein, with the two electrons located in a "charge transfer" complex between flavoprotein and pyridine nucleotide.     

Differential scanning calorimetric studies of E. coli aspartate transcarbamylase. III. The denaturational thermodynamics of the holoenzyme with single-site mutations in the catalytic chain

Aspartate transcarbamylase (EC 2.1.3.2) from E. coli is a multimeric enzyme consisting of two catalytic subunits and three regulatory subunits whose activity is regulated by subunit interactions. Differential scanning calorimetric (DSC) scans of the wild-type enzyme consist of two peaks, each comprised of at least two components, corresponding to denaturation of the catalytic and regulatory subunits within the intact holoenzyme (Vickers et al., J. Biol. Chem. 253 (1978) 8493; Edge et al., Biochemistry 27 (1988) 8081). We have examined the effects of nine single-site mutations in the catalytic chains. Three of the mutations (Asp-100-Gly, Glu-86-Gln, and Arg-269-Gly) are at sites at the C1: C2 interface between c chains within the catalytic subunit. These mutations disrupt salt linkages present in both the T and R states of the molecule (Honzatko et al., J. Mol. Biol. 160 (1982) 219; Krause et al., J. Mol. Biol. 193 (1987) 527). The remainder (Lys-164-Ile, Tyr-165-Phe, Glu-239-Gln, Glu-239-Ala, Tyr-240-Phe and Asp-271-Ser) are at the C1: C4 interface between catalytic subunits and are involved in interactions which stabilize either the T or R state. DSC scans of all of the mutants except Asp-100-Gly and Arg-269-Gly consisted of two peaks. At intermediate concentrations, Asp-100-Gly and Arg-269-Gly had only a single peak near the Tm of the regulatory subunit transition in the holoenzyme, although their denaturational profiles were more complex at high and low protein concentrations. The catalytic subunits of Glu-86-Gln, Lys-164-Ile and Asp-271-Ser appear to be significantly destabilized relative to wild-type protein while Tyr-165-Phe and Tyr-240-Phe appear to be stabilized. Values of delta delta G degree cr, the difference between the subunit interaction energy of wild-type and mutant proteins, evaluated as suggested by Brandts et al. (Biochemistry 28 (1989) 8588) range from -3.7 kcal mol-1 for Glu-86-Gln to 2.4 kcal mol-1 for Tyr-165-Phe.     

Crystallographic and biochemical studies of the (inactive) Lys-49 phospholipase A2 from the venom of Agkistridon piscivorus piscivorus.

Chemical, genetic, and structural studies have defined a critical role for Asp-49 in the calcium-mediated activation of extracellular phospholipases A2 (PLA2). In 1984, a new class of PLA2 was isolated in which this invariant aspartate was replaced with a lysine (Maragnore, J.M., Merutka, G., Cho, W., Welches, W., Kezdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843; Maragnore, J.M., and Heinrikson, R.L. (1986) J. Biol. Chem. 261, 4797-4804). The enzymatic activity of Lys-49 PLA2s has been questioned based on biochemical, mutational, and structural studies (van den Bergh, C.J., Slotboom, A.J., Verheij, H.M., and de Haas, G.H. (1988) Eur. J. Biochem. 176, 353-357). In this paper, we describe the structures of two crystal forms of the Lys-49 PLA2 isolated from the venom of Agkistridon piscivorus piscivorus. The refined models, along with complementary biochemical analysis, clarify the structural basis for the enzymatic inactivity of Lys-49 proteins.     

Modification of synthetic peptides related to lactate dehydrogenase (231-242) by protein carboxyl methyltransferase and tyrosine protein kinase: effects of introducing an isopeptide bond between aspartic acid-235 and serine-236

The possibility that isoaspartyl residues contribute to the substrate specificity of eucaryotic protein carboxyl methyltransferases and/or tyrosine protein kinases has been investigated with two synthetic oligopeptides, Lys-Gln-Val-Val-Asp/isoAsp-Ser-Ala-Tyr-Glu-Val-Ile-Lys, which correspond to amino acids 231-242 of lactate dehydrogenase. One version of the peptide contains the normal amino acid sequence of the chicken muscle M4 isozyme. The other version contains an isoaspartyl residue in position 235 in place of the normal aspartyl residue; i.e., Asp-235 is linked to Ser-236 via its side-chain beta-carboxyl group, rather than via the usual alpha-carboxyl linkage. The normal peptide corresponds to the sequence around Tyr-238 that is phosphorylated in Rous sarcoma virus infected chick embryo fibroblasts [Cooper, J. A., Esch, F. S., Taylor, S. S., & Hunter, T. (1984) J. Biol Chem. 259, 7835]. Using protein carboxyl methyltransferase purified from bovine brain, we found that the normal peptide did not serve as a methyl-accepting substrate but that the isopeptide served as an excellent substrate, exhibiting a stoichiometry of one methyl group per peptide and Km of 0.54 microM. With tyrosine protein kinase partially purified from normal rat spleen both peptides were found to serve as phosphate acceptors at Tyr-238, exhibiting Km values of 4.7 and 8.9 mM for the normal and isopeptide versions, respectively. These results support the idea that protein carboxyl methyltransferase selectively methylates the alpha-carboxyl group of atypical isoaspartyl residues. In contrast, the presence of isoaspartate had a modest negative effect on substrate activity for a tyrosine protein kinase from rat spleen.     

The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. The amino acid sequences of the smaller cyanogen bromide fragments

Fourteen fragments have been isolated from hemocyanin component II of Limulus polyphemus by cleavage with CNBr. The amino acid sequences of the two largest fragments, CNBr Ia and Ib, have been determined (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749; Behrens, P. Q., Nakashima, H., Yokota, E., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). We have determined the amino acid sequence of the remaining 12 smaller fragments.     

Adrenodoxin reductase and adrenodoxin. Mechanisms of reduction of ferricyanide and cytochrome c.

Adrenodoxin reductase, the flavoprotein moiety of the adrenal cortex mitochondrial steroid hydroxylating system, participates in adrenodoxin-dependent cytochrome c and adrenodoxin-independent ferricyanide reduction, with NADPH as electron donor for both of these 1-electron reductions. For ferricyanide reduction, adrenodoxin reductase cycles between oxidized and 2-electron-reduced forms, reoxidation proceeding via the neutral flavin (FAD) semiquinone form (Fig. 9). Addition of adrenodoxin has no effect upon the kinetic parameters of flavoprotein-catalyzed ferricyanide reduction. For cytochrome c reduction, the adrenodoxin reductase-adrenodoxin 1:1 complex has been shown to be the catalytically active species (Lambeth, J. D., McCaslin, D. R., and Kamin, H. (1976) J. Biol. Chem. 251, 7545-7550). Present studies, using stopped flow techniques, have shown that the 2-electron-reduced form of the complex (produced by reaction with 1 eq of NADPH) reacts rapidly with 1 eq of cytochrome c (k approximately or equal to 4.6 s-1), but only slowly with a second cytochrome c (k = 0.1 to 0.3 s-1). However, when a second NADPH is included, two more equivalents of cytochrome are reduced rapidly. Thus, the adrenodoxin reductase-adrenodoxin complex appears to cycle between 1- and 3-electron reduced states, via an intermediate 2-electron-containing form produced by reoxidation by cytochrome (Fig. 10). For ferricyanide reduction by adrenodoxin reductase, the fully reduced and semiquinone forms of flavin each transfer 1 electron at oxidation-reduction potentials which differ by approximately 130 mV. However, adrenodoxin in a complex with adrenodoxin reductase allows electrons of constant potential to be delivered from flavin to cytochrome c via the iron sulfur center...     

The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. The amino acid sequence of the second largest cyanogen bromide fragment

Fourteen fragments have been isolated from hemocyanin component II of Limulus polyphemus by cleavage with CNBr. The amino acid sequence of the largest fragment, CNBr Ia has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequence of the 12 smaller fragments is reported in an accompanying paper (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1985) J. Biol. Chem. 261, 10511-10519). We have determined the amino acid sequence of the second largest fragment, CNBr Ib. The fragment contains 142 residues and has a molecular weight of 16,095.     

The oxidation of apomorphine and other catechol compounds by horseradish peroxidase: relevance to the measurement of dihydropteridine reductase activity

It has been reported by Shen et al. (Shen, R.-S., Smith, R.V., Davis, P.J. and Abell, C.W. (1984) J. Biol. Chem. 259, 8894-9000) that apomorphine and dopamine are potent, non-competitive inhibitors of quinonoid dihydropteridine reductase. In this paper we show that apomorphine, dopamine and other catechol-containing compounds are oxidized rapidly to quinones by the horseradish peroxidase-H2O2 system which is used to generate the quinonoid dihydropterin substrate. These quinones react non-enzymatically with reduced pyridine nucleotides, depleting the other substrate of dihydropteridine reductase. When true initial rates of dihydropteridine reductase-dependent reduction of quinonoid dihydropterins are measured, neither apomorphine nor any other catechol-containing compound that has been tested has been found to inhibit dihydropteridine reductase.     

Molecular determinants by which a long chain toxin from snake venom interacts with the neuronal alpha 7-nicotinic acetylcholine receptor

Long chain curarimimetic toxins from snake venom bind with high affinities to both muscular type nicotinic acetylcholine receptors (AChRs) (K(d) in the pm range) and neuronal alpha 7-AChRs (K(d) in the nm range). To understand the molecular basis of this dual function, we submitted alpha-cobratoxin (alpha-Cbtx), a typical long chain curarimimetic toxin, to an extensive mutational analysis. By exploring 36 toxin mutants, we found that Trp-25, Asp-27, Phe-29, Arg-33, Arg-36, and Phe-65 are involved in binding to both neuronal and Torpedo (Antil, S., Servent, D., and Ménez, A. (1999) J. Biol. Chem. 274, 34851-34858) AChRs and that some of them (Trp-25, Asp-27, and Arg-33) have similar binding energy contributions for the two receptors. In contrast, Ala-28, Lys-35, and Cys-26-Cys-30 selectively bind to the alpha 7-AChR, whereas Lys-23 and Lys-49 bind solely to the Torpedo AChR. Therefore, alpha-Cbtx binds to two AChR subtypes using both common and specific residues. Double mutant cycle analyses suggested that Arg-33 in alpha-Cbtx is close to Tyr-187 and Pro-193 in the alpha 7 receptor. Since Arg-33 of another curarimimetic toxin is close to the homologous alpha Tyr-190 of the muscular receptor (Ackermann, E. J., Ang, E. T. H., Kanter, J. R., Tsigelny, I., and Taylor, P. (1998) J. Biol. Chem. 273, 10958-10964), toxin binding probably occurs in homologous regions of neuronal and muscular AChRs. However, no coupling was seen between alpha-Cbtx Arg-33 and alpha 7 receptor Trp-54, Leu-118, and Asp-163, in contrast to what was observed in a homologous situation involving another toxin and a muscular receptor (Osaka, H., Malany, S., Molles, B. E., Sine, S. M., and Taylor, P. (2000) J. Biol. Chem. 275, 5478-5484). Therefore, although occurring in homologous regions, the detailed modes of toxin binding to alpha 7 and muscular receptors are likely to be different. These data offer a molecular basis for the design of toxins with predetermined specificities for various members of the AChR family.     

Cadmium-substituted skeletal troponin C metal binding investigations and sequence assignment of the cadmium-113 resonances

The binding of cadmium to the calcium binding subunit of skeletal troponin (STnC) has been reinvestigated using direct binding methods and fluorescent derivatives. These data provide straightforward explanations of the observed titration behavior in the 113Cd NMR (Ellis, P.D., Strang, P., and Potter, J.D. (1984) J. Biol. Chem. 259, 10348-10356). Further, fluorescent derivatives of skeletal troponin C provide an excellent means of establishing a sequence assignment for the resonances observed in the 113Cd NMR. The results of these experiments demonstrate that sites I and II, the Ca2+ regulatory sites, can be assigned to resonances at -108.5 and -101.5 ppm, respectively. Sites III and IV, the structural sites, are assigned to resonances -112.8 and -106.8 ppm, respectively. These data are discussed in terms of recent structural findings and speculations.     

DNA sequences from the adenovirus 2 genome

The sequence of 5,839 nucleotides from the adenovirus 2 genome has been determined and includes the regions between coordinates 32-44% and 66-71%. These regions contain the coding sequences for the 52,55K polypeptide, polypeptide IIIa, penton base, and the N terminus of the 100K polypeptide. Several additional unidentified open reading frames are present, including examples which overlap identified reading frames on the complementary strand and on the same strand. In conjunction with previously published sequences and those described in the accompanying papers (Akusj?rvi, G., Alestr?m, P., Pettersson, M., Lager, M., J?urnvall, H., and Pettersson, U. (1984) J. Biol. Chem. 259, 13976-13979; Alestr?m, P., Akusj?rvi, G., Lager, M., Yeh-kai, L., and Pettersson, U. (1984) J. Biol. Chem. 259, 13980-13985) a complete sequence of 35,937 nucleotide pairs can now be reconstructed for the adenovirus 2 genome.     

Structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. Sequences of the overlapping peptides, ordering the CNBr fragments, and the complete amino acid sequence

The amino acid sequence of the largest fragment, CNBr Ia (203 residues) has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequences of the second largest fragment, CNBr Ib (142 residues), and of the 12 smaller fragments are reported in accompanying papers (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10511-10519; Behrens, P. Q., Nakashima, H., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). The complete amino acid sequence of hemocyanin component II has been established by isolation and analysis of 13 methionine-containing peptides from either a tryptic digest or a Staphylococcus aureus strain V8 protease digest of whole carboxamidomethylated hemocyanin II. Hemocyanin II is composed of 628 residues and has a molecular weight with two copper atoms of 72,946.     

Paratope determination of the antithrombotic antibody 82D6A3 based on the crystal structure of its complex with the von Willebrand factor A3-domain

The antithrombotic monoclonal antibody 82D6A3 is directed against amino acids Arg-963, Pro-981, Asp-1009, Arg-1016, Ser-1020, Met-1022, and His-1023 of the von Willebrand factor A3-domain (Vanhoorelbeke, K., Depraetere, H., Romijn, R. A., Huizinga, E., De Maeyer, M., and Deckmyn, H. (2003) J. Biol. Chem. 278, 37815-37821). By this, it potently inhibits the interaction of von Willebrand factor to collagens, which is a prerequisite for blood platelet adhesion to the injured vessel wall at sites of high shear. To fully understand the mode of action of 82D6A3 at the molecular level, we resolved its crystal structure in complex with the A3-domain and fine mapped its paratope by construction and characterization of 13 mutants. The paratope predominantly consists of two short sequences in the heavy chain CDR1 (Asn-31 and Tyr-32) and CDR3 (Asp-99, Pro-101, Tyr-102 and Tyr-103), forming one patch on the surface of the antibody. Trp-50 of the heavy and His-49 of the light chain, both situated adjacent to the patch, play ancillary roles in antigen binding. The crystal structure furthermore confirms the epitope location, which largely overlaps with the collagen binding site deduced from mutagenesis of the A3-domain (Romijn, R. A., Westein, E., Bouma, B., Schiphorst, M. E., Sixma, J. J., Lenting, P. J., and Huizinga, E. G. (2003) J. Biol. Chem. 278, 15035-15039). We herewith further consolidate the location of the collagen binding site and reveal that the potent action of the antibody is due to direct competition for the same interaction site. This information allows the design of a paratope-mimicking peptide with antithrombotic properties.     

Proximity of the catalytic region and the kringle 2 domain in the closed conformer of plasminogen

Introduction of a single intramolecular cross-link with 1,5-difluoro-2,4-dinitrobenzene into Glu-plasminogen freezes the molecule in its closed conformational state (Bányai, L. and Patthy, L. (1984) J. Biol. Chem. 259, 6466-6471). Here we show that the cross-link connects Lys-203 of the kringle 2 domain and Tyr-671 of the catalytic domain, indicating that these regions are in close proximity in the closed conformer of Glu-plasminogen. Comparison of the parameters of the urokinase-catalysed activation of native and cross-linked Glu-plasminogen species indicates that cross-linking of kringle 2 and the catalytic region interferes with the productive binding of urokinase to plasminogen.     

Crystallization and preliminary diffraction studies of the Lys-49 phospholipase A2 from Agkistrodon piscivorus piscivorus

Previous chemical and structural studies have proposed a major role for Asp-49 in the calcium-mediated activation of phospholipases A2. Recently, a new class of phospholipases A2 has been characterized with a lysine in the place of aspartate at position 49 (Maraganore, J. M., Merutka, G., Cho, W., Welches, W., Kézdy, F. J., and Heinrikson, R. L. (1984) J. Biol. Chem. 259, 13839-13843; Maraganore, J. M., and Heinrikson, R. L. (1986) J. Biol. Chem. 261, 4797-4804). Although both the Lys-49 and Asp-49 phospholipases require calcium for enzymatic activity, the Lys-49 enzymes appear to be unique in their ability to bind phospholipids prior to undergoing calcium-mediated activation. We have successfully crystallized the Lys-49 phospholipase A2 from the venom of the American cottonmouth water moccasin (Agkistrodon piscivorus piscivorus). The crystals are tetragonal, the space group being P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 71.05 A, and c = 57.76 A. There is only one molecule in the asymmetric unit and the crystals provide good quality diffraction data to 2.2 A.     

Bilin attachment sites in the alpha, beta, and gamma subunits of R-phycoerythrin. Structural studies on singly and doubly linked phycourobilins

Three unique bilin peptides, a beta subunit peptide bearing a doubly linked phycourobilin (PUB), and two gamma subunit peptides with singly linked PUB groups, were obtained by enzymatic degradation of Gastroclonium coulteri R-phycoerythrin. These peptides were shown to have the sequences (Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260, 4856-4863): (Formula: see text) The sequence of peptide beta-3T was identical to that previously established for a doubly linked phycoerythrobilin (PEB) peptide derived from a B-phycoerythrin (Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M. (1984) J. Biol. Chem. 259, 5472-5480). Secondary ion mass spectrometry of beta-3T yielded a protonated molecular ion of 1629 mass units, the same as that given by the doubly linked PEB peptide (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484), indicating that the doubly linked PUB and PEB tetrapyrroles were isomeric structures. High resolution 1H NMR analyses of peptides beta-3T, gamma-BV8, and gamma-DP provided unambiguous structural assignments for the singly and doubly linked PUB chromophores and indicated that the peptides in gamma-BV8 and gamma-DP were linked to ring A. The determination of which peptide fragment is linked to ring A and which to ring D in peptide beta-3T was not achieved in this study. 1H NMR analyses of three PEB-peptides from G. coulteri R-phycoerythrin--alpha-1 Cys(PEB)-Tyr-Arg, alpha-2 Leu-Cys(PEB)-Val-Pro-Arg, and beta-1 Met-Ala-Ala-Cys(PEB)-Leu-Arg--showed that they were identical to previously described corresponding chromopeptides from Porphyridium cruentum B-phycoerythrin, with the peptide linked to ring A of PEB in each instance (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5485-5489). This is the first documented report on the structure of singly or doubly linked phycourobilins.     

Spectral evidence for tyrosine ionization linked to a conformational change in liver alcohol dehydrogenase ternary complexes.

Resonance energy transfer from Trp-314 to ionized Tyr-286 was proposed (Laws, W. R., and Shore, J. D. (1978) J. Biol. Chem. 253, 8593-8597) as the mechanism for the observed decrease in protein fluorescence of liver alcohol dehydrogenase seen with alkaline pH, or with the formation of a ternary complex with NAD+ and trifluoroethanol. In the present study, ultraviolet difference spectra confirm the presence of ionized tyrosine not only in these two cases but also in the ternary complex with NADH and isobutyramide. Our results indicate that ternary complex formation, with either oxidized or reduced coenzyme, causes a conformational change leading to partial ionization of tyrosine residues in regions of the enzyme far from the active site.     

Ionic effects on adrenal steroidogenic electron transport. The role of adrenodoxin as an electron shuttle.

We have shown (Seybert, D., Lambeth, D., and Kamin, H. (1978), J. Biol. Chem. 253, 8355-8358) that, whereas the 1:1 complex between adrenodoxin reductase and adrenodoxin is the active species for cytochrome c reduction, the complex is not sufficient to allow cytochrome P-45011 beta-mediated hydroxylations;adrenodoxin in excess of reductase is required. In the present studies, reduction by NADPH of excess adrenodoxin is shown to occur at a rate sufficient to support both cytochrome P-450 11 beta-mediated hydroxylation of deoxycorticosterone, and cytochrome P-450sec-mediated side chain cleavage of cholesterol. Oxidation-reduction potential and ion effect studies indicate that the mechanism of steroidogenic electron transport involves an adrenodoxin electron "shuttle" rather than a macromolecular complex of reductase, adrenodoxin, and cytochrome. The oxidation-reduction potential of adrenodoxin is shifted about -100 mV when bound to reductase, and reduction of the iron-sulfur protein thus promotes dissociation of the complex. The rate of adrenodoxin reduction is first stimulated, then inhibited by increasing salt; the effect is ion-specific, with Ca2+ approximately Mg2+ greater than Na+ greater than NH/+. Similar ion-specific rate effects are observed for both of the cytochrome P-450-mediated hydroxylations, indicating that the same reduction mechanism is required for these reactions. Increasing salt concentrations caused dissociation of the complex; dissociation of the form of the complex containing reduced adrenodoxin occurred at lower salt concentrations than that containing oxidized adrenodoxin. The order of effectiveness of ions in causing dissociation is the same as the order for stimulation of adrenodoxin reduction, suggesting a dissociation step in the mechanism. This proposed model, together with dissociation constants for the form of the complex containing either oxidized or reduced adrenodoxin, allows accurate prediction of the salt rate effects curve. For all ions, an activity maximum is seen at the ion concentration which produces the largest molar difference between associated-oxidized and dissociated-reduced states, and the model predicts the positions of the maxima for adrenodoxin reduction, 11 beta-hydroxylation, and side chain cleavage. Thus reduction-induced dissociation of adrenodoxin from adrenodoxin reductase appears to be a required step in steroidogenic electron transport by this system, and a role for adrenodoxin as a mobile electron shuttle is proposed.     

The lysine-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. Relation of structure and function to other phospholipases A2

A new class of phospholipases A2 that have a lysine at position 49 differ from the more conventional Asp-49 enzymes with respect to the sequential binding of the essential cofactor, calcium, and the substrate, phospholipid, in the formation of the catalytic complex (Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kézdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843). We report here the complete amino acid sequence of the Lys-49 enzyme from Agkistrodon piscivorus piscivorus. The sequence was determined by automated Edman degradation of the intact, S-carboxymethylcysteinyl protein and of peptides derived therefrom by cleavage with cyanogen bromide, chymotrypsin, trypsin, and endoproteinase Lys-C. Despite several changes at amino acid residues previously considered to be invariant, the Lys-49 enzymes are homologous to the Asp-49 phospholipases. Homology is especially apparent in the following: 1) the pattern of 14 half-cystine residues, 2) conservation of hydrophobic residues which have been shown to encircle the active site, and 3) conservation of Asp-99 and His-48 which have been implicated in the catalytic reaction itself. These observations together with kinetic and binding data imply that the Lys-49 phospholipases have a catalytic mechanism and a three-dimensional architecture similar to those of the Asp-49 enzymes. Modeling of the Lys-49 enzyme based upon the structure of bovine pancreatic phospholipase reveals that the epsilon-amino group of Lys-49 can fit easily in the calcium-binding site and, moreover, that this orientation of a cationic side chain at position 49 could account for the characteristic and novel feature of the Lys-49 phospholipases, i.e. that they are able to form complexes with phospholipid in the absence of calcium.     

Kinetic and ultraviolet spectroscopic studies of active-site mutants of delta 5-3-ketosteroid isomerase

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes the highly efficient isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position, via an enolic intermediate. An acidic residue responsible for the protonation of the 3-carbonyl function of the steroid and a basic group concerned with the proton transfer have been implicated in the catalytic mechanism. Recent NMR studies with a nitroxide spin-labeled substrate analogue have allowed positioning of the steroid into the 2.5-A X-ray crystal structure of the enzyme [Kuliopulos, A., Westbrook, E.M., Talalay, P., & Mildvan, A.S. (1987) Biochemistry 26, 3927-3937], thereby corroborating the approximate location of the steroid binding site deduced from a difference Fourier X-ray diffraction map of the 4-(acetoxymercuri)estradiol-isomerase complex [Westbrook, E.M., Piro, O.E., & Sigler, P.B. (1984) J. Biol. Chem. 259, 9096-9103]. The steroid lies in a hydrophobic cavity near Asp-38, Tyr-14, and Tyr-55. In order to assess the role of these amino acid residues in catalysis, the gene for isomerase was cloned, sequenced, and overexpressed in Escherichia coli [Kuliopulos, A., Shortle, D., & Talalay, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8893-8897], and the following mutants were prepared: Asp-38 to asparagine (D38N) and Tyr-14 and Tyr-55 to phenylalanine (Y14F and Y55F, respectively). The kcat value of the D38N mutant enzyme is 10(5.6)-fold lower than that of the wild-type enzyme, suggesting that Asp-38 functions as the base which abstracts the 4 beta-proton of the steroid in the rate-limiting step. Threefold lower Km values in all mutants indicate tighter binding of the substrate to the more hydrophobic sites. In comparison with the wild-type enzyme, the Y55F mutant shows only a 4-fold decrease in kcat while the Y14F mutant shows a 10(4.7)-fold decrease in kcat, suggesting that Tyr-14 is the general acid. The red shift of the ultraviolet absorption maximum of the competitive inhibitor 19-nortestosterone from 248 to 258-260 nm, which occurs upon binding to the wild-type enzyme [Wang, S.F., Kawahara, F.S., & Talalay, P. (1963) J. Biol. Chem. 238, 576-585], is mimicked in strong acid. This spectral shift was also observed with the D38N and Y55F mutants, but not on binding of the steroid to the Y14F mutant.(ABSTRACT TRUNCATED AT 400 WORDS)