Protoporphyrinogen IX oxidase from Plasmodium falciparum is anaerobic and is localized to the mitochondrion

Earlier studies in this laboratory had shown that the malarial parasite can synthesize heme de novo and inhibition of the pathway leads to death of the parasite. It has been proposed that the pathway for the biosynthesis of heme in Plasmodium falciparum is unique involving three different cellular compartments, namely mitochondrion, apicoplast and cytosol. Experimental evidences are now available for the functionality and localization of all the enzymes of this pathway, except protoporphyrinogen IX oxidase (PfPPO), the penultimate enzyme. In the present study. PfPPO has been cloned, expressed and shown to be localized to the mitochondrion by immunofluorescence microscopy. Interestingly, the enzyme has been found to be active only under anaerobic conditions and is dependent on electron transport chain (ETC) acceptors for its activity. The native enzyme present in the parasite is inhibited by the ETC inhibitors, atovaquone and antimycin. Atovaquone, a well known inhibitor of parasite dihydroorotate dehydrogenase, dependent on the ETC, inhibits synthesis of heme as well in P. falciparum culture. A model is proposed to explain the ETC dependence of both the pyrimidine and heme-biosynthetic pathways in P. falciparum. (C) 2010 Elsevier B.V. All rights reserved.

Interactions of methoxyamine with pyridoxal-5'-phosphate-Schiff's base at the active site of sheep liver serine hydroxymethyltransferase

The mechanism of interaction of methoxyamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in enzyme activity, visible absorption spectra, circular dichroism and fluorescence, and by evaluating the rate constant by stopped-flow spectrophotometry. Methoxyamine can be considered as the smallest substituted aminooxy derivative of hydroxylamine. It was a reversible noncompetitive inhibitor (Ki = 25 microM) of SHMT similar to O-amino-D-serine. Like in the interaction of O-amino-D-serine and aminooxyacetic acid, the first step in the reaction was very fast. This was evident by the rapid disappearance of the enzyme-Schiff base absorbance at 425 nm with a rate constant of 1.3 x 10(3) M-1 sec-1 and CD intensity at 430 nm. Concomitantly, there was an increase in absorbance at 388 nm (intermediate I). The next step in the reaction was the unimolecular conversion (1.1 x 10(-3) sec-1) of this intermediate to the final oxime absorbing at 325 nm. The identity of the oxime was established by its characteristic fluorescence emission at 460 nm when excited at 360 nm and by high performance liquid chromatography. These results highlight the specificity in interactions of aminooxy compounds with sheep liver serine hydroxymethyltransferase and that the carboxyl group of the inhibitors enhances the rate of the initial interaction with the enzyme.

Modes of binding of guanosine monophosphates to ribonuclease T1 - A computer modelling study

Computer-modelling studies on the modes of binding of the three guanosine monophosphate inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to ribonuclease (RNase) T1 have been carried out by energy minimization in Cartesian-coordinate space. The inhibitory power was found to decrease in the order 2'-GMP > 3'-GMP > 5'-GMP in agreement with the experimental observations. The ribose moiety was found to form hydrogen bonds with the protein in all the enzyme-inhibitor complexes, indicating that it contributes to the binding energy and does not merely act as a spacer between the base and the phosphate moieties as suggested earlier. 2'-GMP and 5'-GMP bind to RNase T1 in either of the two ribose puckered forms (with C3'-endo more favoured over the C2'-endo) and 3'-GMP binds to RNase T1 predominantly in C3'-endo form. The catalytically important residue His-92 was found to form hydrogen bond with the phosphate moiety in all the enzyme-inhibitor complexes, indicating that this residue may serve as a general acid group during catalysis. Such an interaction was not found in either X-ray or two-dimensional NMR studies.

Studies on simultaneous nhibition of trypsin and chymotrypsin by horsegram Bowman-Birk inhibitor

Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of the known three-dimensional structures of BBIs revealed similarities and subtle differences.The hydrophobic core, deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI, binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.

Regulation of the activity of mung bean (Phaseolus aureus) glutamine synthetase by amino acids and nucleotides

The activity of glutamine synthetase isolated from the germinated seedlings of Phaseolus aureus was regulated by feedback inhibition by alanine, glycine, histidine, AMP, and ADP. When glutamate was the varied substrate, alanine, histidine, and glycine were partial noncompetitive, competitive, and mixed-type inhibitors, respectively. The type of inhibition by these amino acids was confirmed by fractional inhibition analysis. The adenine nucleotides, AMP and ADP, completely inhibited the enzyme activity and were competitive with respect to ATP. Multiple inhibition analyses revealed the presence of separate and nonexclusive binding sites for the amino acids and mutually exclusive sites for adenine nucleotides. Cumulative inhibition was observed with these end products.

Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5'-Phosphate- Dependent Enzyme

Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5'-phosphate-containing enzyme, but the requirement of pyridoxal-5'-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of serine hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5'-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of pyridoxal-5'-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with L-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5'-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5'-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-D-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-D-serine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5'-phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean serine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibiton by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyridoxal-5'-phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean serine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5'-phosphate that is present in the mammalian and bacterial enzymes.

Plant aspartate transcarbamylase: An affinity chromatographic method for the purification of the enzyme from germinated seedlings

A simple and rapid affinity chromatographic method for the isolation of aspartate transcarbamylase from germinated seedlings of mung bean (Phaseolus aureus) was developed. A partially purified preparation of the enzyme was chromatographed on an affinity column containing aspartate linked to CNBr-activated Sepharose 4B. Aspartate transcarbamylase was specifically eluted from the column with 10 mImage aspartate or 0.5 Image KCl. The enzyme migrated as a single sharp band during disc electrophoresis at pH 8.6 on polyacrylamide gels. Electrophoresis of the sodium dodecyl sulfate-treated enzyme showed two distinct protein bands, suggesting that the mung bean aspartate transcarbamylase was made up of nonidentical subunits. Like the enzyme purified by conventional procedures, this enzyme preparation also exhibited positive homotropic interactions with carbamyl phosphate and negative heterotropic interactions with UMP. This method was extended to the purification of aspartate transcarbamylase from Lathyrus sativus, Eleucine coracona, and Trigonella foenum graecum.

DNA Polymerase4 from the Silk Glands of Bombyx mori

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3' --> 5' exonuclease activity which participates in proofreading by mismatch match repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.

Medetomidiinin glukuronidaation inhibitio

Lääkeainemetabolialla tarkoitetaan entsymaattisia reaktioita, jotka muuttavat lääkeaineita paremmin elimistöstä poistuvaan muotoon. Lääkeaineet voivat vaikuttaa toistensa metaboliaan inhiboimalla tai indusoimalla metaboloivia entsyymejä. Tällaisten interaktioiden seurauksena lääkeaineen pitoisuus elimistössä voi kasvaa jopa toksiseksi tai vähentyä merkittävästi. Tämä on erityisesti ongelmana silloin, kun käytössä on useita lääkkeitä samanaikaisesti. Lääketutkimuksessa onkin keskitytty tällaisten interaktioiden ennustamiseen ja niitä yritetään välttää tai ainakin vähentää. Työssä tutkittiin medetomidiinia, jonka on äskettäin havaittu metaboloituvan UDP-glukuronosyylitransferaasien (UGT) välityksellä. Työn tarkoituksena oli löytää medetomidiinin glukuronidaatiota inhiboivia yhdisteitä. Lisäksi haluttiin selvittää mahdollisen inhibition mekanismeja. On yleistä tutkia tietyn entsyymin substraatin interaktioita muiden saman perheen entsyymien kanssa. On kuitenkin harvinaisempaa tutkia tällaisia interaktioita kahden eri entsyymiperheen välillä. Tässä työssä tutkittiin inhiboivatko mahdolliset sytokromi P450 -entsyymiä (CYP) inhiboivat yhdisteet myös medetomidiinia glukuronoivia UDP-glukuronosyylitransferaaseja. Glukuronidaation inhibitiota tutkittiin HPLC-menetelmällä, joka on kehitetty aiemmin medetomidiinin glukuronidaation tutkimiseen. Aluksi glukuronidaatiota tutkittiin ilman inhibiittoreita. Tämän jälkeen tutkittiin kolmen mahdollisen inhibiittoriyhdisteen vaikutuksia medetomidiinin glukuronidaatioon ja tuloksia verrattiin ilman inhibiittoria saatuihin tuloksiin. Kolmen tutkitun yhdisteen havaittiin inhiboivan medetomidiinin glukuronidaatiota. Tutkimuksessa havaittiin myös mielenkiintoinen ilmiö, jossa inhibiittoriyhdisteen sitoutuminen aiheutti entsyymikineettisiä muutoksia UDP-glukuronosyylitransferaasin toiminnassa. On mielenkiintoista, että samat yhdisteet inhiboivat sekä CYP- että UGT-metaboliaa. Tulosten perusteella voidaan päätellä, että jos CYP ja UGT metaboloivat samaa yhdistettä, on mahdollista että yhdisteen rakenteelliset analogit aiheuttavat interaktioita molempien entsyymien kanssa. Uusia lääkeaineita kehitettäessä onkin otettava huomioon yleisesti tunnettujen CYP-entsyymien lisäksi myös UGT:t ja niiden mahdolliset yhteisvaikutukset.