Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes.

We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria. Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism. This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties. Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis. The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K. This latter loop is present only in type II citrate synthase sequences. Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network. Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer. The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases. This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation. Another surprising observation from the structure of type II E. coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site. Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur. Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure. Thus, acetylcoenzyme A binding to type II E. coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite. We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases. Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.

Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques.

We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.

Structural and spectroscopic studies of azide complexes of horse heart myoglobin and the His-64-->Thr variant.

The high-resolution X-ray crystallographic structures of horse heart azidometmyoglobin complexes of the wild-type protein and the His-64-->Thr variant have been determined to 2.0 and 1.8 A respectively. Azide binds to wild-type metmyoglobin in a bent configuration with an Fe-N-1-N-3 angle of 119 degrees and is oriented into the distal crevice in the direction of Ile-107. The proximity of the His-64 NE2 atom to the N-1 atom of the bound azide indicates stabilization of the ligand by the His-64 side chain through hydrogen bonding. In addition, structural characterization of wild-type horse heart azidometmyoglobin establishes that the only structural change induced by ligand binding is a small movement of the Leu-29 side chain away from the azide ligand. EPR and Fourier transform infrared spectroscopy were used to characterize the myoglobin azide complexes further. EPR spectroscopy revealed that, in contrast with wild-type azidometmyoglobin, two slightly different low-spin species are formed by azide bound to the His-64-->Thr variant both in solution and in a polycrystalline sample. One of these low-spin species has a greater relative intensity, with g values very similar to those of the azide complex of the wild-type protein. These EPR results together with structural information on this variant indicate the presence of two distinct conformations of bound azide, with one form predominating. The major conformation is comparable to that formed by wild-type myoglobin in which azide is oriented into the distal crevice. In the minor conformation the azide is oriented towards the exterior of the protein.

A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket.

Well-ordered internal amino acids can contribute significantly to the stability of proteins. To investigate the importance of the hydrophobic packing interface between helices G and H in the proximal heme pocket of horse heart myoglobin, the highly conserved amino acid, Leu104, was substituted with asparagine, a polar amino acid of similar size. The Leu104Asn mutant protein and its recombinant wild-type horse heart myoglobin counterpart were expressed from synthetic genes in Escherichia coli. Thermal denaturation of these two recombinant myoglobins, as studied by measurement of circular dichroism ellipticity at 222 nm, revealed that the Leu104Asn mutant had a significantly lower t(m) (71.8 +/- 1 degree C, pH 7.0) than recombinant wild-type myoglobin (81.3 +/- 1 degree C, pH 7.0). To examine the extent to which this 10 degrees C decrease in thermal stability was associated with structural perturbations, X-ray diffraction techniques were used to determine the three-dimensional structures of both the recombinant wild-type and Leu104Asn myoglobins to 0.17 nm resolution. Refinement of these structures gave final crystallographic R-factors of 16.0% and 17.9%, respectively. Structural comparison of the natural and recombinant wild-type myoglobins, together with absorption spectroscopic and electron paramagnetic resonance (EPR) analyses, confirmed the proper expression and folding of the recombinant protein in E. coli. Surprisingly, despite the decreased thermal stability of the Leu104Asn mutant, there are no significant structural differences between the mutant and wild-type myoglobins. EPR and absorption spectroscopic analyses further confirmed the similar nature of the heme iron centres in both proteins. Thus, the introduction of an energetically unfavourable change in side chain polarity at position 104 into a hydrophobic environment that does not support the hydrogen bonding potential of the mutant asparagine appears to perturb important stabilizing helix-helix and heme-protein interactions. The induced structural destabilization is thereby reflected by a significant decrease in the t(m) of horse heart myoglobin.

Electrostatic modification of the active site of myoglobin: characterization of the proximal Ser92Asp variant.

The structural and functional consequences of the introduction of a negatively charged amino acid into the active site of horse heart myoglobin have been investigated by replacement of the proximal Ser92 residue (F7) with an aspartyl residue (Ser92Asp). UV-visible absorption maxima of various ferrous and ferric derivatives and low-temperature EPR spectra of the metaquo (metMb) derivative indicate that the active site coordination geometry has not been perturbed significantly in the variant. 1H-NMR spectroscopy provides direct evidence for the existence of a distal water molecule as the sixth ligand in the oxidized form of the variant at pD 5.7. Spectrophotometric pH titration of the Ser92Asp variant is consistent with this finding and with a pKa = 8.90 +/- 0.02 [25.0 degrees C, mu = 0.10 M (NaCl)] for titration of the distal water molecule, identical to the value reported for the wild-type protein. X-ray crystallography of the metMb derivative indicates that the heme substituents conserve their orientations in the variant protein, except for a slight reorientation of the pyrrole A propionate group to which Ser92 normally hydrogen bonds and reorientation of the carboxyl end of the pyrrole D propionate group. No change is observed in conformation of the proximal (His93) or distal (Wat156) heme ligands. 1H-NMR spectroscopy of the metMbCN form of the protein indicates that a slight rotation of the proximal His93 ligand has occurred in this derivative. Resonance Raman experiments indicate increased conformational heterogeneity in the proximal pocket of the variant. Failure to detect electron density for the Asp residue in the X-ray diffraction map of the variant protein and high average thermal factors for the pyrrole A propionate substituent are consistent with this observation. The variant exhibits novel pH-dependent behavior in the metMb form, as shown by 1H-NMR spectroscopy, and provides evidence for a heme-linked titratable group with a pKa of 5.4 in this derivative. The metMbCN and deoxyMb derivatives also exhibit pH-dependent behavior, with pKas of 5.60 +/- 0.07 and 6.60 +/- 0.07, respectively, compared to the wild-type values of 5.4 +/- 0.04 and 5.8 +/- 0.1. The heme-linked ionizable group is proposed to be His97 in all three derivatives. The reduction potential of the variant is 72 +/- 2 mV vs SHE [25.0 degrees C, mu = 0.10 M (phosphate), pH 6.0], an increase of 8 mV over the wild-type value. The possible influence of a number of variables on the magnitude of the reduction potential in myoglobin and other heme proteins is discussed.

Dobutamine stress echocardiography in the evaluation of late anthracycline cardiotoxicity in childhood cancer survivors.

Late anthracycline cardiotoxicity has been of increasing concern to pediatric oncologists. An increasing number of patients with cardiac dysfunction has been reported without a good correlation between cardiac function or symptoms and routine echocardiographic follow-up. We studied dobutamine stress echocardiography in patients who had received moderate doses of anthracyclines years before. Twenty-three patients (14 male, 9 female; 7-25 y) who completed chemotherapy with moderate doses of anthracyclines (180-380 mg/m2) more than 2 y previously underwent dobutamine stress echocardiography and were compared with a control group of 26 healthy young people (15 male, 11 female; 6-26 y) matched for age and weight. Dobutamine was administered in three periods up to a rate of 5 micro g/kg/min. Eighty-five percent of the patients showed an abnormal response to dobutamine. Both systolic and diastolic functions were affected. The systolic dysfunction was not related to diminished contractility but to an elevated systolic wall stress due to inadequate cardiac muscle thickening. The diminished wall thickening was related to the length of follow-up. Dobutamine proved to be a very sensitive method to detect clinical and subclinical cardiac dysfunction in patients post anthracycline chemotherapy and questions the concept of a safe dose.

Origin of the pH-dependent spectroscopic properties of pentacoordinate metmyoglobin variants.

The pH dependence of the electronic and EPR spectra of two variants of horse heart myoglobin (Mb) in which the distal His64 ligand has been replaced by either Thr or Ile has been studied. Both of these variants exhibit spectroscopic changes with pH that are indicative of a transition between two ferric high-spin forms that occurs with a pKa of 9.49 for the His64Thr variant and 9.26 for the His64Ile variant and that is distinctly different from the pH-dependent spectroscopic changes related to titration of the distal aquo ligand of wild-type Mb. The electronic and EPR spectra of both variants at all values of pH studied are consistent with the presence of a pentacoordinate heme iron center. For the His64Thr variant, a high-resolution (1.9 A) structure determination establishes the lack of the distal aquo ligand and demonstrates an out-of-plane movement of the ferric iron toward the proximal histidine together with a decrease of the Fe-His bond length. Investigation of this pH-linked equilibrium by EPR spectroscopy reveals rhombically split high-spin signals at both pH 7 and 11 with a greater degree of rhombicity exhibited by the alkaline species. We propose that the pH-linked spectroscopic transition exhibited by these distal histidine variants results from the deprotonation of the proximal His93 residue to produce imidazolate ligation at alkaline pH.

The proximal ligand variant His93Tyr of horse heart myoglobin.

The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for bovine liver catalase. The 1D 1H-NMR spectrum of the variant confirms the five-coordinate nature of the heme iron center and exhibits a broad resonance at 112.5 ppm that is attributable to the meta protons of the phenolate ligand. This result indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is unchanged from that of the wild-type protein (pH 8) while the midpoint reduction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 mV lower. The three-dimensional structure of the variant determined by X-ray diffraction analysis confirms the five-coordinate nature of the heme iron center and establishes that the introduction of a proximal Tyr ligand is accommodated by a shift of the F helix (residues 88-99) in which this residue resides away from the heme pocket. Additional effects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hydrogen bonding interactions with the heme prosthetic group.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone metabolism and mineralisation after cytotoxic chemotherapy including ifosfamide.

Lumbar spine bone mineral density and bone mineral metabolism were studied in 13 children three months or more after completion of cytotoxic chemotherapy that included ifosfamide given for different malignancies. Blood and urine were analysed for calcium, phosphorus, and magnesium and blood for alkaline phosphatase activity, parathyroid hormone, and 1,25(OH)2 vitamin D3. Bone mineral density (BMD) was measured at the lumbar spine (L1-L4) using a commercial dual x ray absorptiometer. Serum concentrations of calcium, phosphorus, and magnesium and alkaline phosphatase activity, as well as plasma 1,25(OH)2 vitamin D3 concentrations were normal in all children. Slightly raised parathyroid hormone concentrations were seen in two children. An increased urinary excretion of calcium was found in five children. Mean (SD) BMD of the children was -0.88 (1.44). Three children had osteopenia, as defined by a BMD lower than -2 SD for age and sex related standards. No significant relation was found between the BMD and the biochemical parameters. In conclusion, a normal BMD was found in most patients who had received ifosfamide, even in those with persisting hypercalciuria.

Prevention of emesis by tropisetron (Navoban) in children receiving cytotoxic therapy for solid malignancies.

Emesis is one of the most frequent and distressing adverse effects of cytotoxic chemotherapy. Conventional antiemetic regimens are unsatisfactory due to their poor efficacy and their adverse events, particularly in children. Tropisetron (Navoban; Sandoz Pharma Ltd, Basel, Switzerland) belongs to a new class of 5-hydroxytryptamine receptor antagonists with antiemetic effectiveness in patients receiving anticancer agents. We evaluated tropisetron (0.2 mg/kg/d) in 24 chemotherapy-treated children who had experienced severe emesis during previous chemotherapy courses in spite of concomitant administration of either alizapride or metoclopramide. Complete control of emesis was achieved in 70% of the courses (37% of those including cisplatin). No severe adverse effect was reported. Headache was observed in two courses and constipation was observed during two other courses. Tropisetron proved clearly superior to conventional antiemetics and safe in use.

FTIR analysis of the interaction of azide with horse heart myoglobin variants.

The interaction of azide with variants of horse heart myoglobin (Mb) has been characterized by Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and UV-VIS absorption spectroscopy and by molecular modeling calculations. Distal histidine variants (His64Thr, His64Ile, His64Lys) and charged surface variants (Val67Arg, Lys45Glu, Lys45Glu/Lys63Glu) were included in this study. All variants, with the exception of Val67Arg, have a lower azide affinity than the wild-type protein. Analysis of the temperature dependence of the FTIR spectra (277-313 K) revealed that the wild-type protein and all variants exhibit a high-spin/low-spin equilibrium. Introduction of positively charged amino acid residues shifts nu max for the low-spin form to higher energy while negatively charged residues shifted this maximum to lower energy. The low azide binding affinity exhibited by the His64Thr and His64Ile variants is accompanied by a shift of the nu max for the low-spin infrared band to lower energy and by a significant increase in the corresponding half-bandwidths. This observation indicates greater mobility of the bound azide ligand in these variants. The His64Lys variant exhibits two infrared bands attributable to low-spin forms that are assigned to two different conformations of the lysyl residue. In one conformation, the lysine is proposed to form a hydrogen bond with the bound azide similar to that proposed to occur between the distal histidine and bound azide, and in the other conformation no interaction occurs.

Structural characterization of heme ligation in the His64-->Tyr variant of myoglobin.

A site-specific mutant of horse heart myoglobin has been prepared in which the distal heme pocket residue, His64, is replaced by tyrosine. The structure of this myoglobin variant has been determined to 2.0-A resolution using x-ray diffraction techniques and refined to a final crystallographic R-factor of 16.9%. The polypeptide backbone conformation of the His64-->Tyr variant of myoglobin is very similar to that of the wild-type protein. However, in the variant the water normally found coordinated to the heme iron atom and hydrogen-bonded to His64 has been displaced by the hydroxyl oxygen of the Tyr64 side chain. The tyrosine oxygen atom is directly coordinated to the heme iron atom with a bond length of 2.18 A. Distortion of heme planarity and changes in the packing of the Leu29 and Leu104 side chains are related to this mutation. The ligand environment of the ferric iron has been studied by electron paramagnetic resonance (EPR) spectroscopy using crystalline material and protein in solution. The protein in solution exhibits a rhombically split ferric high spin EPR spectrum with g values of 6.64, 5.34, and 1.98. The EPR spectrum of the crystalline sample consists of two different ferric high spin signals. The main signal is similar to the signal observed in solution and is assigned to His93-Fe(III)-Tyr64 coordination. The relatively high rhombicity of this signal can be explained as arising from distortions of the heme plane seen in the crystal structure. The second, more axial high spin signal found in the crystalline state can be tentatively assigned to another form of iron ligation with a different iron-tyrosine bond length and a less distorted heme plane.

Prevention of emesis by ICS 205-930 in children receiving cytotoxic chemotherapy.

Nausea and vomiting are among the most frequent and severe acute side-effects of cytotoxic therapy and are not optimally controlled by conventional antiemetics. This situation warrants the evaluation of new classes of antiemetic agents such as the 5-HT3 receptor antagonists. 19 children with a median age of 9 years (range 2-16 years), treated with cytotoxic drug combinations that had previously caused nausea and vomiting refractory to conventional antiemetics, were given the selective 5-HT3 receptor antagonist ICS 205-930. The drug was given intravenously (i.v.) at 0.2 mg/kg (maximum 5 mg) during the chemotherapy infusion period and was continued orally for up to 5 days in chemotherapy courses containing cisplatin. The number of emetic episodes was recorded and the response was scored according to following scale: grade 1 = no nausea, no emetic episode; grade 2 = up to four episodes of vomiting and less than 5 h of nausea; grade 3 = five or more than five emetic episodes and/or nausea for at least 5 h. The 19 patients received a total of 169 various courses of chemotherapy combined with ICS 205-930. A score of 3 was observed during one course only, a score of 2 in 37 out of the 169 courses, including the four courses with cisplatin. The drug was very well tolerated. Side-effects possibly related to ICS 205-930 were mild to moderate headache in 4 patients during seven courses overall and obstipation in 3 patients during 11 courses. The results strongly suggest that ICS 205-930 is a highly effective and safe antiemetic agent in non-naive pediatric patients receiving non-cisplatin cytotoxic chemotherapy and who had failed conventional antiemetic treatment.

Neuroblastoma today.

Notwithstanding the progress made in radiology, nuclear medicine, immunohematology and genetics for more accurate diagnosis and staging in neuroblastoma and the availability of general new efficacious cytostatic drugs, the prognosis of children over 1 yr with advanced disease has remained poor. New refinements in therapeutics with multiagent regimens, massive chemotherapy followed by autologous bone marrow transplantation treatment, with or without immunomagnetic purging, and/or total body irradiation have improved response rate and disease-free survival in metastatic patients, but their effect on long-term survival needs further evaluation.

Melanotic neuroectodermal tumor of infancy with high serum levels of alpha-fetoprotein. Ultrastructural study and immunological evidence of glial fibrillary protein and alpha-fetoprotein.

We report an infant melanotic neuroectodermal tumor in the anterior part of the upper gingiva, which was associated with high urinary excretion of vanilmandelic acid. Serum levels of alpha-fetoprotein were abnormally high (200 ng/ml). They returned to normal after removal of the tumor (10 ng/ml). The tumor was characterized histologically and ultrastructurally by the presence of melanocytic and astrocytic cells containing glial fibrillary protein. Intracellular alpha-fetoprotein was demonstrated by the immunoperoxidase method (PAP) in scattered foci of interstitial cells.

Treatment of acute lymphoblastic leukemia in children with the BFM protocol. A cooperative pilot study.

From January 1981 through July 1983, 141 children with newly diagnosed acute lymphoblastic leukemia were registered in a cooperative clinical study whose objective was to evaluate the toxicity and the feasibility of the German Berlin-Frankfort-Münster (BFM) protocol. The results were comparable with those reported by the BFM group. For the 133 patients (94%) who achieved complete remission, the actuarial disease-free survival was 67% at 4 years. These results were obtained in spite of a high rate of deaths in complete remission during the initial year of the study. Subsequently, probably as a result of improved expertise in the handling of the protocol, the proportion of toxic deaths declined sharply. Although the current BFM protocol adjusts for aggressiveness of therapy according to the volume of the liver and spleen, splenomegaly (but not hepatomegaly) remained of prognostic significance. Moreover, for patients with bad risk features, an initial high hemoglobin level was found to represent an additional factor of negative significance.