Mutagenesis and Laue structures of enzyme intermediates: isocitrate dehydrogenase

Site-directed mutagenesis and Laue diffraction data to 2.5 A resolution were used to solve the structures of two sequential intermediates formed during the catalytic actions of isocitrate dehydrogenase. Both intermediates are distinct from the enzyme-substrate and enzyme-product complexes. Mutation of key catalytic residues changed the rate determining steps so that protein and substrate intermediates within the overall reaction pathway could be visualized.     

Crystal structures of reaction intermediates of L-2-haloacid dehalogenase and implications for the reaction mechanism

Crystal structures of L-2-haloacid dehalogenase from Pseudomonas sp. YL complexed with monochloroacetate, L-2-chlorobutyrate, L-2-chloro-3-methylbutyrate, or L-2-chloro-4-methylvalerate were determined at 1.83-, 2.0-, 2.2-, and 2.2-A resolutions, respectively, using the complex crystals prepared with the S175A mutant, which are isomorphous with those of the wild-type enzyme. These structures exhibit unique structural features that correspond to those of the reaction intermediates. In each case, the nucleophile Asp-10 is esterified with the dechlorinated moiety of the substrate. The substrate moieties in all but the monochloroacetate intermediate have a D-configuration at the C2 atom. The overall polypeptide fold of each of the intermediates is similar to that of the wild-type enzyme. However, it is clear that the Asp-10-Ser-20 region moves to the active site in all of the intermediates, and the Tyr-91-Asp-102 and Leu-117-Arg-135 regions make conformational changes in all but the monochloroacetate intermediates. Ser-118 is located near the carboxyl group of the substrate moiety; this residue probably serves as a binding residue for the substrate carboxyl group. The hydrophobic pocket, which is primarily composed of the Tyr-12, Gln-42, Leu-45, Phe-60, Lys-151, Asn-177, and Trp-179 side chains, exists around the alkyl group of the substrate moiety. This pocket may play an important role in stabilizing the alkyl group of the substrate moiety through hydrophobic interactions, and may also play a role in determining the stereospecificity of the enzyme. Moreover, a water molecule, which is absent in the substrate-free enzyme, is present in the vicinities of the carboxyl carbon of Asp-10 and the side chains of Asp-180, Asn-177, and Ala-175 in each intermediate. This water molecule may hydrolyze the ester intermediate and its substrate. These findings crystallographically demonstrate that the enzyme reaction proceeds through the formation of an ester intermediate with the enzyme's nucleophile Asp-10.     

The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates

The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.     

Electron paramagnetic resonance studies of cobalt-substituted angiotensin I-converting enzyme

The traditional treatment of high-flow vascular malformations consists of selective embolization, surgical removal, or a combination of both. Recurrence of the lesion and bleeding control are still the main problems, and the result of treatment is sometimes disappointing. We suggest treatment of these lesions with surgical ligation of the distal major feeding arteries followed by intravascular injection of a sclerosing agent (3% tetradecyl sulfate), and surgical excision and reconstruction when indicated. We have found this to be an effective treatment regimen. We present 14 cases of high-flow vascular malformations of the head and neck area treated with this approach, of which 4 cases developed skin necrosis. Three of these 4 cases of skin necrosis were later treated with skin grafting and, in 1 case, an upper arm skin tube flap was used for nasal tip reconstruction. Three cases underwent delayed reconstruction using tissue expanders. From a symptomatic and aesthetic point of view, preliminary satisfactory results were obtained. We feel that this approach is a good option for treating difficult, high-flow vascular malformations.     

Electron paramagnetic resonance and dynamic nuclear polarization of char suspensions: surface science and oximetry

Carbon chars have been synthesized in our laboratory from a variety of starting materials, by means of a highly controlled pyrolysis technique. These chars exhibit electron paramagnetic resonance (EPR) line shapes which change with the local oxygen concentration in a reproducible and stable fashion; they can be calibrated and used for oximetry. Biological stability and low toxicity make chars good sensors for in vivo measurements. Scalar and dipolar interactions of water protons at the surfaces of chars may be utilized to produce dynamic nuclear polarization (DNP) of the 1H nuclear spin population in conjunction with electron Zeeman pumping. Low-frequency EPR, DNP and DNP-enhanced MRI all show promise as oximetry methods when used with carbon chars.     

Electron microscopic evidence for the mechanism of blood-retinal barrier breakdown in diabetic rabbits: comparison with magnetic resonance imaging

Diabetes leads to a breakdown of the blood-retinal barrier (BRB), which can be demonstrated in experimental models by immunocytochemistry and magnetic resonance imaging (MRI). The present study utilizes these methods to investigate the mechanism of BRB breakdown in diabetic rabbits, a model ideally suited to both procedures. Rabbits were treated with alloxan and examined 2 months, 1 year, and 1.5 years after the development of diabetes to assess BRB breakdown using MRI and immunocytochemical staining for endogenous albumin. Using MRI, an increased incidence of retinal vascular leakage is first evident at 1 year of diabetes. Electron microscopic immunolocalization of albumin suggests that BRB compromise is principally mediated by transendothelial transport of serum proteins in endocytic vesicle-like structures of approximately 0.4-1 micron diameter. Some additional retinal vascular leakage is occasionally demonstrated through the interendothelial cell tight junctions, but only when adjacent vascular endothelial cells show degenerative changes. The similarity of these findings to those previously reported for diabetic humans and rats supports the use of the diabetic rabbit as a model for studying BRB dysfunction. MRI and electron microscopic (EM) immunocytochemistry are complementary methods for evaluating BRB dysfunction. MRI can provide an overall picture of the entire eye without sacrificing the animal. EM immunocytochemistry can provide a more detailed picture of a limited area of interest to gain insight into the mechanisms of extravasation. Together, both methods provide a more complete understanding of BRB breakdown in diabetic rabbits.     

pKa measurements from nuclear magnetic resonance for the B1 and B2 immunoglobulin G-binding domains of protein G: comparison with calculated values for nuclear magnetic resonance and X-ray structures

Two-dimensional homo- and heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine pKa values for all of the acidic residues in the B1 and B2 immunoglobulin G- (IgG-) binding domains of protein G. Due to the stability of protein G over a wide pH range, estimates of ionization constants were also obtained for some basic residues. These experimentally determined ionization constants were compared with values calculated from both X-ray and NMR-derived structures of B1 and B2 using the UHBD algorithm [Antosiewicz, J., et al. (1994) J. Mol. Biol. 238, 415-436]. This algorithm has been found to be predictive for pKa measurements in proteins and, in combination with experimental measurements, allowed some evaluation of the NMR and X-ray structures. Three regions where significant differences exist between the X-ray and NMR structures are (1) the position of the E56 side chain relative to the backbone amides of K10 and D40, (2) residues 33-37 in the helix, and (3) the Y45 side-chain conformation. For all three cases, the experimental pH titration curves are notably more consistent with the X-ray structures than the NMR structures. In contrast, a number of solvent-accessible side chains have experimental pKas more in agreement with mean pKas calculated from families of NMR structures. The conformations of these side chains may be susceptible to crystal packing effects. From titration experiments under basic conditions, it is noteworthy that the chemical shift of the Y45 C epsilonH resonance is invariant up to pDcorr 12. The Y45 side-chain hydroxyl group appears to maintain a nativelike hydrogen bond with D47 at pDcorr 12, even though the protein is approximately 90% unfolded. These results suggest that this short-range (i, i + 2) interaction, located in the beta3-beta4 hairpin, is present in the high-pH denatured state and may therefore form early in the folding of protein G.     

Urinary excretion of cyclophosphamide in humans, determined by phosphorus-31 nuclear magnetic resonance spectroscopy

Phosphorus-31 NMR spectroscopy was used to analyze urine samples from patients treated with cyclophosphamide (CP) on 2 consecutive days. CP and all of its known phosphorylated metabolites except the tautomeric pair 4-hydroxycyclophosphamide/aldophosphamide, i.e. carboxycyclophosphamide (CXCP), dechloroethylcyclophosphamide (DCCP), alcophosphamide, ketophosphamide, and phosphoramide mustard (PM), were determined. Several other signals corresponding to unknown CP-related compounds were observed. Seven of them were identified; all were hydrolysis products of CP or its metabolites (one from CP, two from CXCP, three from DCCP, and one from PM). Twenty-four-hour urinary excretion of unmetabolized CP was not significantly different on the first (17% of the daily administered dose) and second (16%) days of treatment. The amounts of phosphorylated metabolites excreted in 24-hr urine samples were much higher after the second CP dose (37%) than after the first (20%), suggesting autoinduction of CP metabolism. CXCP and its two degradation products (accounting for 7-10% of CXCP) were by far the major metabolites (11.5 and 23% after the first and second doses, respectively). DCCP plus its degradation products and alcophosphamide each represented 2-3% on the first day of treatment and 5% on the second day of treatment. Levels of PM and its degradation products were extremely low (0.3 and 0.6% after the first and second CP doses, respectively), as were those of ketophosphamide (0.4 and 0.6% on the first and second days of treatment, respectively). We noted only modest interpatient variation in excreted levels of CP and all of its metabolites.     

Hydroperoxidase activities of ferrihemes: heme analogues of peroxidase enzyme intermediates

By reaction of deuteroferriheme (DFH) with peroxo acids, spectroscopically distinct and peroxidatically active deuteroferriheme-peroxide compounds (DPC) are formed. These species closely resemble, and are probably identical with, the species formed by reaction of DFH with H2O2, which has previously been considered to be an analogue of peroxidase compound I. Stopped-flow spectrophotometric titration studies imply: (a) that DPC is formed by reaction of 1.9 +/- 0.2 mol of DFH with 1 mol of peroxo acid; (b) that the spectral change accompanying formation of DPC is independent of the peroxo acid oxidant used. Titration of the oxidizing power of DPC formed with H2O2 implies that submaximal yields of DPC are obtained, a result that could implicate DPC species as analogues of catalase compound I in the catalase action of ferrihemes. Preliminary results suggest that DPC may involve both monomeric and dimeric heme components.     

Radiation-induced radical formation in beta-glycerophosphate single crystals studied by electron paramagnetic resonance and electron nuclear double resonance spectroscopy: formation of an allylic-type radical

The purpose of this study was to better understand students' perceptions of and preferences for inclusion or pull-out service delivery models. Thirty-two students with and without learning disabilities who had participated in both models during the past 2 or 3 years were interviewed individually. Key questions assessed their perceptions of which model was most conducive to academic learning and which was most likely to yield social benefits, and the reasons for their beliefs. Results indicated that students' views varied. Overall, more children identified pull-out as the model of choice, but many children were confident that inclusion was meeting their academic and social needs. We interpret the results of this study as providing support for maintaining a continuum of service delivery options and for considering the placement of each child individually, based on his or her unique needs.     

Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy

Nephrocalcin (NC) is a calcium-binding glycoprotein of 14,000 molecular weight. It inhibits the growth of calcium oxalate monohydrate crystals in renal tubules. The NC used in this study was isolated from bovine kidney tissue and purified with the use of DEAE-cellulose chromatography into four isoforms, designated as fractions A-D. They differ primarily according to the content of phosphate and gamma-carboxy-glutamic acid. Fractions A and B are strong inhibitors of the growth of calcium oxalate monohydrate crystal, whereas fractions C and D inhibit crystal growth weakly. Fraction A, with the highest Ca(2+)-binding affinity, was characterized with respect to its metal-binding sites by using the vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies. By EPR spectrometric titration, it was shown that fraction A of NC bound VO2+ with a stoichiometry of metal:protein binding of 4:1. Also, the binding of VO2+ to NC was shown to be competitive with Ca2+. Only protein residues were detected by proton ENDOR as ligands, and these ligands bound with complete exclusion of solvent from the inner coordination sphere of the metal ion. This type of metal-binding environment, as derived from VO(2+)-reconstituted NC, differs significantly from the binding sites in other Ca(2+)-binding proteins.     

Mutational destabilization of the critical interface water cluster in Scapharca dimeric hemoglobin: structural basis for altered allosteric activity

We determined the proportion of survival variability explained by the usual prognostic factors in childhood acute lymphoblastic leukaemia (ALL) during a prognostic study of 1552 patients enrolled in three consecutive Fralle group protocols (Fralle 83, Fralle 87 and Fralle 89). The event-free survival rates at 5 years were 54.8% (SD 1.9), 43.1%) (SD 2.7) and 55.6% (SD 2.2), respectively. In the univariate analysis the following variables were predictive of poor outcome: male gender, elevated leucocytosis (> 50 x 10(9)/l), circulating blastosis. haemoglobin >12 g/dl, platelet count <100 x 10(9)/l, age under 1 year or over 9 years, enlarged mediastinum, nodes, spleen and liver, T phenotype, absence of CD10+ cells; testicular and meningeal involvement, poor response to induction therapy (CCSG M3), and LDH >400 U/l. Among the cytogenetic features, hyperdiploidy had a protective effect, whereas hypodiploidy, translocation and other structural abnormalities had a negative influence, particularly in cases of t(9;22) or t(4;11). Multivariate analysis summarized the prognostic information in terms of four variables: age, gender, leucocytosis and cytogenetic features. Missing data had little influence on the results. However, despite their significance in the multivariate analysis, these four variables each had very low predictive power (1.1% for gender, 2.0% for age, 3.5% for leucocytosis, and 1.6% for cytogenetic features). Thus, the most significant prognostic factors in childhood ALL each explain no more than 4% of the variability in prognosis. This may explain the disappointing practical value of these factors and underlines the need for prognostic tools in childhood ALL.     

A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies

Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.     

Structure effects of double D-amino acid replacements: a nuclear magnetic resonance and circular dichroism study using amphipathic model helices

D-Amino acid replacements and the determination of resulting structural changes are a useful tool to recognize amphipathic helices in biologically active peptides such as neuropeptide Y and corticotropin-releasing factor. In this paper the secondary structures of one amphipathic alpha-helical peptide and its double D-amino acid analog have been determined by means of 1H NMR and CD spectroscopies under equivalent conditions. The chemical shifts (NH and C alpha H) and the analysis of nuclear Overhauser effects show a split of the continuous helix for the all-L peptide into two helices at the position of double D-amino acid replacement. Hydrogen exchange rates correlate with water accessibilities in the hydrophobic/hydrophilic face and confirm the amphipathic helical structure in the all-L peptide as well as in its double D-amino acid analog. A significantly accelerated hydrogen isotope exchange rate is observed for the D-Ala9 backbone proton, implying an increased flexibility at that position. These results show that the incorporation of an adjacent pair of D-amino acids only causes a local change in structure and flexibility, which makes the double D replacement interesting as a tool for specific helix-disturbing modifications to search for helical conformations in biologically active peptides.