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S Englard

Publications and source records attributed to S Englard.

17 recordsLinked to original sources
PubMed1991

Cosubstrate binding site of Pseudomonas sp. AK1 gamma-butyrobetaine hydroxylase. Interactions with structural analogs of alpha-ketoglutarate.

Forty-one aromatic and aliphatic analogs of alpha-ketoglutarate were studied kinetically for their interaction with the alpha-ketoglutarate binding site of gamma-butyrobetaine hydroxylase obtained from Pseudomonas sp. AK1. Together, the compounds represent structural permutations probing the contribution of: 1) the C5 carboxyl group of alpha-ketoglutarate (domain I); 2) the C1-C2 keto acid moiety of alpha-ketoglutarate (domain II); 3) the distance between domains I and II; and 4) the spatial relationship of the two domains required for optimal interaction with the cosubstrate binding site. All compounds were competitive inhibitors for alpha-ketoglutarate (Km 0.018 mM). Functionally, two subsites of the cosubstrate binding site were evident: subsite I for polar interaction with the C5 carboxyl group, and subsite II, comprising of two distinct cis-oriented coordination sites of the catalytic ferrous ion which interact with the C1-C2 keto acid moiety. The most efficient inhibitors were pyridine 2,4-dicarboxylate (Ki 0.0002 mM) and 3,4-dihydroxybenzoate (Ki 0.0006 mM). Both compounds contain a carboxyl group and a chelating moiety corresponding to domains I and II of alpha-ketoglutarate, respectively. The fixed orientation of these groups in both analogs was used to assess intersubsite distance and spatial relationship required for optimal interaction with the cosubstrate binding site. Binding at subsite I and chelation at subsite II were indispensible for effective competitive inhibition. The distance between these two domains also helped determine whether attachment at the cosubstrate binding site would be catalytically productive. This was emphasized by the failure of either oxaloacetate or alpha-ketoadipinate to promote hydroxylation. Optimal interdomain distance, however, was not sufficient for cosubstrate utilization, as pyridine 2,4-dicarboxylate, with an interdomain distance identical to alpha-ketoglutarate in its staggered conformation, did not sustain hydroxylation. In the overall, these studies suggest that alpha-ketoglutarate utilization occurs in a ligand reaction at the active site ferrous ion of gamma-butyrobetaine hydroxylase. This is of particular interest since the delineated stereochemical mode of oxidative decarboxylation could generate the reactive oxo-iron species that was shown experimentally to promote gamma-butyrobetaine hydroxylation by an abstraction-recombination mechanism (Blanchard, J. S., and Englard, S. (1983) Biochemistry 22, 5922-5928; Englard, S., Blanchard, J. S., and Midelfort, C. F. (1985) Biochemistry 24, 1110-1116).

Binding Sites↗
PubMed1990

Reexamination of the 1H NMR assignments and solution conformations of L-carnitine and O-acetyl-L-carnitine using C-2 stereospecifically labeled monodeuterated isomers.

The two C-2 monodeuterated isomers of L-carnitine were synthesized by enzymatic hydration of crotonobetaine in D2O and by enzymatic proton exchange of L-[2-2H2]carnitine in H2O. These reactions, catalyzed by an induced Escherichia coli carnitine hydrolyase proceed stereospecifically. The two isomers of L-[2-2H]carnitine were examined by 1H NMR at 500 MHz, which allowed us to independently monitor the pD dependence and coupling constants of the H-2 protons. The results obtained indicate that there is little effect of the carboxyl charge on the conformational state(s) of L-carnitine about the C-2/C-3 bond. The NMR data obtained in this study do not support previous solution studies of the pH-dependent conformational changes for DL-carnitine nor the proposed conformation of O-acetyl-DL-carnitine in the crystalline state.

Acetylcarnitine↗
PubMed1988

Effect of phi X C protein on leading strand DNA synthesis in the phi X174 replication pathway.

The influence of the bacteriophage phi X174 (phi X) C protein on the replication of bacteriophage phi X174 DNA has been examined. This small viral protein, which is required for the packaging of phi X DNA into proheads, inhibits leading strand DNA synthesis. The inhibitory effect of the phi X C protein requires a DNA template bearing an intact 30-base pair (bp) phi X origin of DNA replication that is the target site recognized by the phi X A protein. Removal of nucleotides from the 3' end of this 30-bp conserved origin sequence prevents the inhibitory effects of the phi X C protein. Leading strand replication of supercoiled DNA substrates containing the wild-type phi X replication origin results in the production of single-stranded circular DNA as well as the formation of small amounts of multimeric and sigma structures. These aberrant products are formed when the termination and reinitiation steps of the replication pathway reactions are skipped as the replication fork moves through the origin sequence. Replication carried out in the presence of the phi X C protein leads to a marked decrease in these aberrant structures. While the exact mechanism of action of the phi X C protein is not clear, the results presented here suggest that the phi X C protein slows the movement of the replication fork through the 30-bp origin sequence, thereby increasing the fidelity of the termination and reinitiation reactions. In keeping with the requirement for the phi X C protein for efficient packaging of progeny phi X DNA into proheads, the phi X C protein-mediated inhibition of leading strand synthesis is reversed by the addition of proteins essential for phi X bacteriophage formation. Incubation of plasmid DNA substrates bearing mutant 30 base pair phi X origin sequences in the complete packaging system results in the in vitro packaging and production of infectious particles in a manner consistent with the replication activity of the origin under study.

Bacteriophage phi X 174↗
PubMed1986

Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus.

In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.

Antibodies, Monoclonal↗
PubMed1986

Trans regulation of the phosphoenolpyruvate carboxykinase (GTP) gene, identified by deletions in chromosome 7 of the mouse.

Livers from newborn mice homozygous for either one of the lethal deletions c14CoS or c3H in chromosome 7 have drastically reduced levels of cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] activity when compared with normal littermates. The structural gene for the enzyme maps on chromosome 2 and appears intact and not grossly rearranged in deletion homozygotes. These mice also have negligible levels of hepatic mRNA encoding this enzyme. Studies of the transcription rate of the gene showed that it was reduced to 25-50% of normal in hepatic nuclei obtained from mice homozygous for either deletion. We suggest that, in addition to the reduction in the level of transcription, the deletions in chromosome 7 may also cause alterations in messenger stability, processing, or transport from the nucleus.

Animals↗
PubMed1986

Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Saccharomyces species.

Three unlinked, homologous genes, STA1, STA2, and STA3, encode the extracellular glycosylated glucoamylase isozymes I, II, and III, respectively, in Saccharomyces species. S. cerevisiae, which is sta0 (absence of functional STA genes in haploids), does carry a glucoamylase gene, delta sta, expressed only during sporulation (W. J. Colonna and P. T. Magee, J. Bacteriol. 134:844-853, 1978; I. Yamashita and S. Fukui, Mol. Cell. Biol. 5:3069-3073, 1985). In this study we examined some of the physiological and genetic factors that affect glucoamylase expression. It was found that STA2 strains grown in synthetic medium produce glucoamylase only in the presence of either Maltrin M365 (a mixture of maltooligosaccharides) or starch. Maximal levels of glucoamylase activity were found in cells grown in rich medium supplemented with glycerol plus ethanol, starch, or Maltrin. When various sugars served as carbon sources they all supported glucoamylase synthesis, although at reduced levels. In any given growth medium glucoamylase isozyme II synthesis was modulated by functionality of the mitochondria. Synthesis of glucoamylase is continuous throughout the growth phases, with maximal secretion taking place in the early stationary phase. In the various regimens, the differences in enzyme accumulation are accounted for by differences in the levels of glucoamylase mRNA. Both glucoamylase mRNA and enzyme activity were drastically and coordinately inhibited in MATa/MAT alpha diploids and by the presence of the regulatory gene STA10. Both effects were partially overcome when the STA2 gene was present on a multicopy plasmid. The STA2 mRNA and glucoamylase were coinduced in sporulating STA2/STA2 diploids. A smaller, coinduced RNA species was also detected by Northern blotting with a STA2 probe. The same mRNA species was detected in sporulating sta0 diploids and is likely to encode the sporulation-specific glucoamylase.

Culture Media↗
PubMed1978

A simplified method for the measurement of gamma-butyrobetaine hydroxylase activity.

A method for the assay of gamma-butyrobetaine hydroxylase activity is described. The procedure is based on the measurement of 3H2O formed from [2,3-3H]gamma-butyrobetaine. The formation of 3H2O was essentially linear with time of incubation and enzyme concentration. Despite a significant isotope effect that causes the extent of hydroxylation to be underestimated, an appropriately determined correction factor permits one to relate quantitatively the degree of detritiation to the amount of carnitine formed. The assay is simple, rapid, specific, accurate, highly reproducible, and relatively sensitive. Its reliability and convenience represent an improvement over existing methods based on the tedious and time-consuming enzymatic radioisotopic determination of the carnitine formed or on the coupled decarboxylation of [1-14C]alpha-ketoglutarate, a method that cannot be used in crude extracts.

Animals↗