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PubMed2006

A monofunctional and thermostable prephenate dehydratase from the archaeon Methanocaldococcus jannaschii.

Prephenate dehydratase (PDT) is an important but poorly characterized enzyme that is involved in the production of L-phenylalanine. Multiple-sequence alignments and a phylogenetic tree suggest that the PDT family has a common structural fold. On the basis of its sequence, the PDT from the extreme thermophile Methanocaldococcus jannaschii (MjPDT) was chosen as a promising representative of this family for pursuing structural and functional studies. The corresponding pheA gene was cloned and expressed in Escherichia coli. It encodes a monofunctional and thermostable enzyme with an N-terminal catalytic domain and a C-terminal regulatory ACT domain. Biophysical characterization suggests a dimeric (62 kDa) protein with mixed alpha/beta secondary structure elements. MjPDT unfolds in a two-state manner (Tm = 94 degrees C), and its free energy of unfolding [DeltaGU(H2O)] is 32.0 kcal/mol. The purified enzyme catalyzes the conversion of prephenate to phenylpyruvate according to Michaelis-Menten kinetics (kcat = 12.3 s-1 and Km = 22 microM at 30 degrees C), and its activity is pH-independent over the range of pH 5-10. It is feedback-inhibited by L-phenylalanine (Ki = 0.5 microM), but not by L-tyrosine or L-tryptophan. Comparison of its activation parameters (DeltaH(++)= 15 kcal/mol and DeltaS(++)= -3 cal mol-1 K-1) with those for the spontaneous reaction (DeltaH(++) = 17 kcal/mol and DeltaS(++)= -28 cal mol-1 K-1) suggests that MjPDT functions largely as an entropy trap. By providing a highly preorganized microenvironment for the dehydration-decarboxylation sequence, the enzyme may avoid the extensive solvent reorganization that accompanies formation of the carbocationic intermediate in the uncatalyzed reaction.

Amino Acid Sequence↗
PubMed1978

Carnitine-acylcarnitine translocase. Inhibition by alpha-cyano-4-hydroxycinnamate and evidence for separate identity from the pyruvate transporting system of mitochondria.

Some of the known inhibitors of pyruvate transport inhibited the activity of carnitine-acylcarnitine translocase. Their order of effectiveness with millimolar concentration required for 50% inhibition given in parentheses, was: Compound UK-5099 (alpha-cyano-beta-(1-phenylindol-3-yl)acrylate) (0.1); alpha-cyano-4-hydroxycinnamate (0.17); alpha-cyano-3-hydroxycinnamate (1); alpha-cyanocinnamate (1); alpha-fluorocinnamate (7); transcinnamate (10); p-hydroxycinnamate (10); phenylpyruvate (22); p-hydroxyphenylpyruvate (25). Kinetically, the alpha-cyano-4-hydroxycinnamate inhibition was mixed and the p-hydroxyphenylpyruvate inhibition was noncompetitive with respect to external (-)-carnitine. The alpha-cyano-4-hydroxycinnamate inhibition was reversible and resulted from its ability to act as a thiol reagent. In general, alpha-cyanocinnamate and its derivatives inhibit carnitine transport at concentrations 100 to 5000 times as high as those known to pyruvate transport. At millimolar concentrations, alpha-cyano-4-hydroxycinnamate inhibited the mitochondrial transport of molecules other than carnitine as well as the activity of carnitine acyltransferases. Pyruvate and carnitine did not complete for transport into and out of mitochondria. These results establish that transmitochondrial transport mechanisms for carnitine and pyruvate involve different carriers.

Acyltransferases↗
PubMed1991

Hexose metabolism in pancreatic islets. Regulation of aerobic glycolysis and pyruvate decarboxylation.

1. D-Glucose (0.5-16.7 mM) preferentially stimulates aerobic glycolysis and D-[3,4-14C]glucose oxidation, relative to D-[5-3H]glucose utilization in rat pancreatic islets, the concentration dependency of such a preferential effect displaying a sigmoidal pattern. 2. Inorganic and organic calcium antagonists, as well as Ca2+ deprivation, only cause a minor decrease in the ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to a high concentration of the hexose (16.7 mM). 3. Non-glucidic nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH), 2-ketoisocaproate and 3-phenylpyruvate fail to stimulate aerobic glycolysis and D-[3,4-14C]glucose oxidation in islets exposed to 6.0 mM D-glucose. Nevertheless, BCH augments [1-14C]pyruvate and [2-14C]pyruvate oxidation. 4. The glucose-induced increment in the paired ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization is impaired in the presence of either cycloheximide or ouabain. 5. These findings suggest that the preferential effect of D-glucose upon aerobic glycolysis and pyruvate decarboxylation is not attributable solely to a Ca(2+)-induced activation of FAD-linked glycerophosphate dehydrogenase and/or pyruvate dehydrogenase, but may also involve an ATP-modulated regulatory process.

Aerobiosis↗
PubMed1992

Study of hexose transport, glycerol phosphate shuttle and Krebs cycle in islets of adult rats injected with streptozotocin during the neonatal period.

At 3-4 degrees C, the transport of 3-O-methyl-D-glucose (30 mM) was severely impaired in islets prepared from adult rats injected with streptozotocin during the neonatal period. However, at 37 degrees C, the first and second phase of glucose-stimulated insulin release were decreased to the same relative extent in perifused islets of diabetic, as compared to control, animals. Moreover, the time-related increase in the oxidative response of the islets to 16.7 mM D-glucose was less pronounced in diabetic than control rats. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase in islet homogenates of diabetic rats only represented one-fifth of that found in control rats, whereas the activity of the cytosolic NAD-glycerophosphate dehydrogenase was comparable in both types of rats. This coincided with the fact that a rise in D-glucose concentration from 2.8 to 16.7 mM failed to increase significantly L-[2-3H]glycerol conversion to 3HOH in islets from diabetic rats, in contrast to the situation found in control animals. The activity of 2-ketoglutarate dehydrogenase in islet homogenates when expressed per microgram protein was not different in control and diabetic rats. Likewise, the ratio between D-[6-14C]glucose oxidation and D-[3,4-14C]glucose oxidation and the capacity of either a non-metabolized analog of L-leucine or 3-phenylpyruvate to preferentially stimulated D-[6-14C]glucose oxidation relative to D-[5-3H]glucose utilization were both unaffected in islets from diabetic rats. These findings argue against the existence of a primary defect in the Krebs cycle of diabetic rats. It is proposed that, despite an obvious alteration of the hexose transport system in the islet cells of diabetic rats, the preferential impairment of the B-cell secretory response to D-glucose, as distinct from other secretagogues, in this model of non-insulin-dependent diabetes is mainly attributable to the low activity of FAD-linked glycerophosphate dehydrogenase, resulting in a decreased metabolic flow through the glycerol phosphate shuttle and a reduced rate of aerobic glycolysis.

3-O-Methylglucose↗
PubMed1975

[Transamination of L-aspartate and L-phenylalanine in Escherichia coli K 12].

At least four separate enzymes are found to catalyze the transamination between phenylalanine and alpha-ketoglutarate in E. coli K 12, one of them being the aspartate aminotransferase. The Km of the latter enzyme for alpha-ketoglutarate is 0.3 or 0.035 mM according to the acceptor aminoacid being phenylalanine or aspartate respectively. The double specificity of aspartate aminotransferase in E. coli is however clearly shown by thermal inactivation studies using various effectors or different temperatures, and by the finding of an active transamination between aspartate and phenylpyruvate in the absence of ketoglutarate. This reaction shows the usual ping-pong type of mechanism, which implies that both substances are substrates for the same protein. Contrary to the phenylalanine-alpha-ketoglutarate reaction, which is probably of little importance in vivo when catalyzed by this enzyme, the direct ketoglutarate-free transamination between aspartate and the aromatic alpha-ketoacid is likely to represent a physiological function in regulating, at least partially, the balance between biosynthetic pathways for aromatic aminoacids and aspartate, for instance by maintaining similar ratios between the aminoacid and its ketoacid partner in both cases. For the sake of clarity it is proposed that the name "transaminase A", first devised by Rudman and Meister, be used for aspartate aminotransferase only, knowing that the specificity of this peculiar enzyme behaves as an accessory agent in the transamination of the aromatic compounds.

Aspartate Aminotransferases↗
PubMed1987

Alpha-ketoacids stimulate rat renal cysteine conjugate beta-lyase activity and potentiate the cytotoxicity of S-(1,2-dichlorovinyl)-L-cysteine.

Renal cysteine conjugate beta-lyase (beta-lyase) catalyzes the bioactivation of nephrotoxic cysteine S-conjugates. beta-Lyase activity is present in both renal cytosolic and mitochondrial fractions, and, although the cytosolic beta-lyase is identical to glutamine transaminase K, the mitochondrial beta-lyase has not been characterized. Because beta-lyase is a pyridoxal phosphate (PLP)-dependent enzyme, pyridoxamine phosphate (PMP) formation may occur during the metabolism of cysteine S-conjugates. In this study, the effects of alpha-ketoacids, which may convert the PMP form of the enzyme to the pyridoxal phosphate form, on the metabolism and cytotoxicity of cysteine S-conjugates were examined; the PMP enzyme is catalytically inactive in beta-elimination reactions, but is catalytically active in transamination reactions. Both alpha-keto-gamma-methiolbutyrate (KMB) and alpha-ketobutyrate enhanced the metabolism of S-(2-benzothiazolyl)-L-cysteine (BTC) to 2-mercaptobenzothiazole by rat renal cytosol or mitochondria. KMB and phenylpyruvate potentiated both the cytotoxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in isolated rat renal proximal tubular cells and the inhibition of mitochondrial respiration produced by DCVC. These results are consistent with the formation of PMP during the renal cytosolic or mitochondrial metabolism of cysteine S-conjugates. Mitochondrial beta-lyase was previously localized in the outer membrane. To examine whether beta-lyase activity is present in mitoplasts, but in the PMP form, the effects of KMB on the metabolism of BTC to 2-mercaptobenzothiazole and on the DCVC-induced inhibition of state 3 respiration in mitoplasts were studied. The majority of the mitochondrial beta-lyase activity was present in the outer membrane, and the specific activity of the outer membrane beta-lyase was greater than that of the mitoplast beta-lyase. KMB produced equivalent stimulation of beta-lyase activity in intact mitochondria, in mitochondrial outer membranes, and in mitoplasts and potentiated DCVC-induced inhibition of respiration in intact mitochondria, but not in mitoplasts. These results provide additional evidence for the central role of beta-lyase in the bioactivation of nephrotoxic cysteine S-conjugates.

Animals↗
PubMed2001

Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis.

cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/L-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and D-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4x10(6) M(-1) x s(-1)) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1x10(4) M(-1) x s(-1)) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of L-dopachrome compared with non-esterified L-dopachrome (>87000-fold) and a high kcat (approximately 4x10(4) s(-1). The crystal structure, determined to 1.65 A (1 A=0.1 nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5 ng/ml-5 pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500 ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6 ng/ml (55 ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.

Amino Acid Sequence↗