Preparation and some properties of stable and carbon-14 and tritium-labeled short-chain aliphatic hydroxamic acids

Fishbein, William N.; Daly, J.C.; Streeter, C. L.

doi:10.1016/0003-2697(69)90152-3

Cited by 67 publications

(14 citation statements)

References 17 publications

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“…Formohydroxamate (m.p. 76-79') was synthesized from ethyl formate by the method of Fishbein et al, (1969). The structures of all synthetic compounds were verified by 'H-NMR.…”

Section: Methodsmentioning

confidence: 99%

Synergistic binding of hydrophobic probes and zinc ligands to thermolysin

Pfuetzner

Chan

1993

European Journal of Biochemistry

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The strong synergism previously observed in the binding of inhibitors to two Zn-proteases, has also been found for thermolysin. As in earlier cases, the effects are produced by a small Zn-ligand (e.g. a hydroxamate) in the presence of another compound which contains the key structural features of specific substrates (a specificity probe). For thermolysin, the most effective specificity probes are hydrophobic derivatives of amines and amino acids (e.g. carbobenzyloxy-L-alaninol). Even the simple combination of benzyl alcohol and formohydroxamate displays considerable synergism. The above effects are temperature dependent and correlate well with a thermally induced conformational isornerization reported recently for this enzyme.Our results seem to be related to previous observations of substrate synergism in the reverse reaction and to superactivation by chemical modification of this enzyme. A11 these effects are consistent with a change in the environment of the catalytically important zinc atom upon binding of the hydrophobic side chain of the substrate. With the inclusion of thermolysin, binding synergism is now known to occur in an endopeptidase as well as in exopeptidases of diverse specificity. The general occurrence of this phenomenon in zinc proteases and its possible significance are discussed in an accompanying study.Previously we reported the synergistic interactions of ligands with microsomal aminopeptidase (MAP ; DiGregorio et al., 1988) and the angiotensin-converting enzyme (ACE) (Pfuetzner and Chan, 1988) which were both Zn-containing proteases. In these studies, the synergism could be detected by following the concurrent effects of two inhibitors on enzyme activity. By means of a suitable graphical analysis (Yonetani and Theorell, 1964), it was possible to determine quantitatively the degree to which binding of one inhibitor increased the affinity of the other towards the enzyme. For both of the above enzymes, synergistic binding was found only between a Zn-ligand and a compound containing key structural features of specific substrates. This structural requirement for synergism was particularly striking because of the large difference in specificity between MAP (Wachsmuth et al., 1966) and ACE (Soffer, 1976). We therefore felt that the above effects might be of some general significance. To probe the extent of the phenomenon, we have broadened our investigation to include thermolysin which is also a Znprotease (for a review see Matthews, 1988). This enzyme has been chosen because it would allow us to determine whether the structural requirements for synergism previously observed for exopeptidases would also apply to an endopeptidase. Another advantage of studying thermolysin is that the

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“…Formohydroxamate (m.p. 76-79') was synthesized from ethyl formate by the method of Fishbein et al, (1969). The structures of all synthetic compounds were verified by 'H-NMR.…”

Section: Methodsmentioning

confidence: 99%

Synergistic binding of hydrophobic probes and zinc ligands to thermolysin

Pfuetzner

Chan

1993

European Journal of Biochemistry

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“…Trypticase was from BBL, and DL-L-methylbutyric acid from Kodak Ltd, Kirkby, Liverpool. Acetohydroxamic acid was prepared as described by Fishbein, Daly & Streeter (1969). All other chemicals were from BDH.…”

Section: Animalsmentioning

confidence: 99%

Urease Activity in the Rumen of Sheep and the Isolation of Ureolytic Bacteria

Cook¹

1976

Journal of General Microbiology

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S U M M A R YUrease activity in the sheep rumen varied with the diet of the sheep, but appeared to be largely or entirely present in the small bacterial fraction. Screening of over 1000 strains of rumen bacteria isolated on different media showed that urease activity was apparently confined to species of Staphylococcus, Lactobacillus casei var. casei and KZebsieZZa aerogenes. Consideration of the numbers in which these occurred and their activities suggested that the bacteria could not be responsible for the total rumen urease activity. By enrichment culture a ureolytic strain of Streptoc o c m faecium was isolated. This had a higher urease activity than the other bacteria and occurred in higher numbers in the rumen. It could live with other bacteria in the rumen of a gnotobiotic lamb in numbers, and with a urease activity, comparable with those in the normal sheep rumen. The other properties of the bacterium also suggested that it would grow and produce urease in the rumen, but was unlikely to retain its urease activity after isolation. It was concluded that this bacterium was the main source of rumen urease in roughage-fed, and probably other, sheep.

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“…The hydroxyurea analogues of Table 1 were obtained from commercial sources (acetohydroxamic acid, formamidoxime and guanazole from EGA Chemie, N-hydroxyguanidine sulfate from Eastman, and N-methylhydroxylamine hydrochloride from Fluka) or synthesized as follows : hydroxyurea [16], formohydroxamic acid [17], N-hydroxyurethane [I 81, benzohydroxamic acid and salicylohydroxamic acid [I 91, glycine hydroxamic acid [20], N-hydroxyoxamide [21] (m.p. 147 OC; anal.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Active Site of Ribonucleotide Reductase of Escherichia coli, Bacteriophage T4 and Mammalian Cells by Inhibition Studies with Hydroxyurea Analogues

Larsen¹,

Sjöoberg²,

Thelander³

1982

European Journal of Biochemistry

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Hydroxyurea specifically inhibits the enzyme ribonucleotide reductase by inactivating the essential tyrosine free radical in one of the two non-identical subunits constituting the enzyme. Studies on the reactivity of hydroxyurea analogues towards the free radical salt, potassium nitrosodisulphonate, and the radical-containing B2 subunit of Escherichia coli ribonucleotide reductase showed that, to obtain maximal enzyme inhibition, the most important parameter of an analogue was the ability to undergo a one-electron oxidation. Also, a common geometrical feature for the analogues with high inhibitory potency was the planarity of the molecules.Results of inhibition studies with analogues containing bulky substituents but having about the same ability to reduce the free radical salt suggested that the tyrosine free radical of protein B2 is located in a pocket about 0.4 nm wide and more than 0.6 nm deep.The ribonucleotide reductases of E. coli, TCinfected E. coli and mammalian cells all contain the same tyrosine free radical structure. Still the mammalian reductase showed a 75-fold higher sensitivity to inhibition by 3,4-dihydroxybenzohydroxamic acid than the E. coli reductase. In contrast, the sensitivity to hydroxyurea was the same for both enzymes. The dihydroxybenzohydroxamic acid and hydroxyurea both reacted immediately (tip < 5 s) with the free radical salt. Therefore the difference in sensitivity between the mammalian and bacterial reductase most probably reflects different topologies of their active sites with the site of the mammalian enzyme being more exposed.The T4-induced reductase showed a 10-fold increased sensitivity towards both hydroxyurea and the polyhydroxybenzohydroxamic acids compared to the E. coli enzyme, indicating a greater ability of the T4 tyrosine free radical to undergo a one-electron reduction. A closer understanding of the different topologies of the active sites of different ribonucleotide reductases could be of great value in the design of target-directed drugs.Ribonucleotide reductase is an allosteric enzyme present in all cells that make D N A and it catalyzes the formation of deoxyribonucleotides from ribonucleotides [I]. In Escherichia coli the enzyme consists of a 1 : 1 complex between two non-identical subunits, proteins B1 and B2, of molecular weights 160000 and 78000 respectively. Protein B1 contains binding sites for the ribonucleoside diphosphate substrates and for the nucleoside triphosphate effectors and it also has oxidation/reduction-active disulfides, which donate the electrons necessary for the reduction. Protein B2 contains nonheme iron and a tyrosine free radical necessary for enzyme activity [2,3]. The active site of the E. coli reductase is known to contain structural elements from both proteins B1 and B2. This was shown in studies using the substrate analogues 2'-chloro-2'-deoxycytidine diphosphate and 2'-azido-2'-deoxycytidine diphosphate, both of which function as highly specific suicidal inhibitors [4].A similar general construction of ribonucfeotide reductase h...

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Preparation and some properties of stable and carbon-14 and tritium-labeled short-chain aliphatic hydroxamic acids

Cited by 67 publications

References 17 publications

Synergistic binding of hydrophobic probes and zinc ligands to thermolysin

Synergistic binding of hydrophobic probes and zinc ligands to thermolysin

Urease Activity in the Rumen of Sheep and the Isolation of Ureolytic Bacteria

Characterization of the Active Site of Ribonucleotide Reductase of Escherichia coli, Bacteriophage T4 and Mammalian Cells by Inhibition Studies with Hydroxyurea Analogues

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