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Stereochemical course of the formation of the C(7)-formyl group from a chiral methyl group during the transformation of chlorophyllide a into chlorophyllide b
No full textThe biosynthesis of chlorophyll a and chlorophyll b from (2R,3R)- and (2S,3S)-5-amino[2,3-14C2,2,3-
2H2,2,3-3H2]levulinic acid in greening barley has established that chlorophyllide a oxidase catalyses the
transformation of the methyl group at C(7) of chlorophyllide a into the CHO group of chlorophyllide b with the
loss of HSi from the 7-(hydroxymethyl)chlorophyllide intermediate
X-Ray Diffraction Images For Human Recombinant 5-Aminolevulinic Acid Dehydratase (Alad).
No full textX-ray diffraction images for recominant human 5-aminolevulinic acid dehydratase (ALAD) collected at ESRF (Grenoble) beam line ID14-2 using an ADSC Quantum 4 detector to a resolution of 2.8 Å. A series of 1 ̊ oscillation images were recorded with an exposure time of 10 seconds per image. More details are given with the scanned notes and the log file. </span
Original X-Ray Diffraction Images For 5-Aminolevulinic Acid Dehydratase (Alad) From E. Coli Complexed With Porphobilinogen.
No full textThe diffraction images which allowed the original 2.1 Angstrom resolution structure determination of Escherichia coli ALAD co-crystallised with a non-covalently bound moiety of the product, porphobilinogen (PBG), are presented. </span
Dimeric pig heart succinate-coenzyme A transferase uses only one subunit to support catalysis
No full textPig heart succinate-coenzyme A transferase (succinyl-coenzyme A: 3-oxoacid coenzyme A transferase; E. C. 2.8.3.5.), a dimeric enzyme purified by affinity chromatography on Procion Blue MX-2G Sepharose, reacts with acetoacetyl-coenzyme A to form a covalent enzyme-coenzyme A thiolester intermediate in which the active site glutamate (E344) of both subunits each forms thiolester links with coenzyme A. Reaction of this dimeric enzyme-coenzyme A species with sodium borohydride leads to inactivation of the enzyme and reduction of the thiolester on both subunits to the corresponding enzyme alcohol, as judged by electrospray mass spectrometry. Reaction of the dimeric enzyme-coenzyme A intermediate with either succinate or acetoacetate, however, results in only one-half of the coenzyme A being transferred to the acceptor carboxylate to form either succinyl-coenzyme A or acetoacetyl-coenzyme A. Reaction of this latter enzyme species with borohydride caused no loss of enzyme activity despite the reduction of the remaining half of the enzyme-coenzyme A thiolester to the enzyme alcohol. That this catalytic asymmetry existed between subunits within the same enzyme dimer was demonstrated by showing that the enzyme species, created by successive reaction with acetoacetyl-coenzyme A and succinate, bound to Blue MX-2G Sepharose through the remaining available active site and could be eluted as a single chromatographic species by succinyl-coenzyme A. It is concluded that while both of the subunits of the succinate-coenzyme A transferase dimer are able to form enzyme-coenzyme A thiolester intermediates, only one subunit is competent to transfer the coenzyme A moiety to a carboxylic acid acceptor to form the new acyl-coenzyme A product. The possible structural basis for this catalytic asymmetry and its mechanistic implications are discussed
Diffraction Images For 5-Aminolevulinic Acid Dehydratase (Alad) From E. Coli Complexed With Porphobilinogen.
No full textThe diffraction images which allowed the 2.1 Angstrom resolution structure determination of Escherichia coli ALAD co-crystallised with a non-covalently bound moiety of the product, porphobilinogen (PBG), are presented. The structure revealed that the pyrrole side chain amino group is datively bound to the active site zinc ion and that the PBG carboxylates interact with the enzyme via hydrogen bonds and salt-bridges with invariant residues. A number of hydrogen bond interactions that were previously observed in the structure of yeast ALAD with a cyclic intermediate resembling the product PBG appear to be weaker in the new structure suggesting that these interactions are only optimal in the transition state. </span
X-Ray Diffraction Images For Human Native 5-Aminolevulinic Acid Dehydratase (Alad).
No full textX-ray diffraction images of human native ALAD collected at station 9.5 at synchrotron radiation source (SRS) Daresbury, UK, with a Marresearch 345 image plate detector on Sunday 26th April 1998. More details of the data collection are given in the files suffixed SUMMARY.</span
Structure and specificity studies on batroxobin, a snake venom derived thrombin-like enzyme
No full textBatroxobin derived from two sources, Bothrops atrox and Bothrops moojeni, two subspecies of the same Bothrops Atrox species, were characterised. It was not possible to identify any difference between them in terms of mass and N-terminal sequence analysis. However, on reaction with various fibrinogens it was evident that the batroxobin derived from Bothrops atrox was twice as active as the batroxobin derived from Bothrops moojeni. Both batroxobins had a preference for fibrinogens derived from rat and mouse plasma, the natural prey of Bothrops Atrox pit vipers.Sequence analysis of the fibrinogens indicated some differences in the sequences around the scissile bond of fibrinopeptide A, which could account for differences observed in specificity between the batroxobins and thrombin. It was found that Batroxobin has an absolute requirement for a proline residue at P2' position, unlike thrombin which can tolerate histidine. It was also evident that batroxobin has a preference to an aspartic acid at the P3 position, whereas thrombin prefers a glycine in this position. The synthesis of a novel inhibitor with a reduced peptide bond showed that both batroxobin and thrombin have a requirement for a carbonyl group at the scissile bond not only for cleavage, but also for recognition.In an attempt to find a structural explanation for the observations, homology modelling was performed. A model produced from the structures of porcine glandular kallikrein and TSV-PA was finally decided upon. The sequences of various peptides were modelled into the active site cleft of the batroxobin model. This work showed that the preference of batroxobin for an aspartic acid at P3 could be due to its ability to form a stabilising interaction with a lysine in the active site cleft, which is not present in thrombin. It also indicated that batroxobin cannot tolerate histidine at the P2' position due to the highly positive nature of the enzyme at that point.</p
The determination of the absolute configurations of diastereomers of (S)- camphanoyl 3-hydroxy-5-oxohexanoic acid derivatives by X-ray crystallography
No full textThis work was supported by the EPSRC, the BBSRC, and by the Wellcome Trust. We also thank the Chemical Database Service provided by the EPSRC at Daresbury. We are grateful to Joan Street for providing the NMR service and Dr. J. Langley for the high resolution mass spectroscopy (HRMS) in the Chemistry Department at Southampton
The X-ray structure of the plant like 5-aminolaevulinic acid dehydratase from Chlorobium vibrioforme complexed with the inhibitor laevulinic acid at 2.6 angstrom resolution
No full text5-Aminolaevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyses the dimerisation of 5-aminolaevulinic acid to form the pyrrole, porphobilinogen. ALAD from Chlorobium vibrioforme is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts a TIM-barrel fold with a 30 residue N-terminal arm extension. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact principally via their arm regions to form octamers in which each active site is located on the surface. The active site contains two invariant lysine residues (200 and 253), one of which (Lys253) forms a Schiff base link with the bound substrate analogue, laevulinic acid. The carboxyl group of the laevulinic acid forms hydrogen bonds with the side-chains of Ser279 and Tyr318. The structure was examined to determine the location of the putative active-site magnesium ion, however, no evidence for the metal ion was found in the electron density map. This is in agreement with previous kinetic studies that have shown that magnesium stimulates but is not required for activity. A different site close to the active site flap, in which a putative magnesium ion is coordinated by a glutamate carboxyl and five solvent molecules may account for the stimulatory properties of magnesium ions on the enzyme.<br/
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