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Biomedical subjects

F Jordan

Publications and source records attributed to F Jordan.

At least 91 records · Page 5Linked to original sources
PubMed1983

Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analogue (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate.

(E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) was found to irreversibly inactivate brewers' yeast pyruvate decarboxylase (PDC, EC 4.1.1.1) in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate. An expression was derived for two-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for two-site binding. The calculated Ki's of 0.7 mM and 0.3 mM for CPB were assigned to the catalytic site and the regulatory site, respectively. The presence of pyruvic acid at high concentrations protected PDC from inactivation, whereas low concentrations of pyruvic acid accelerated inactivation by CPB. Pyruvamide, a known allosteric activator of PDC, was found to enhance inactivation by CPB. The results can be explained if pyruvamide binds only to a regulatory site, but CPB and pyruvic acid compete for both the regulatory and the catalytic centers. [1-14C]CPB was found to lose 14CO2 concurrently with the inactivation of the enzyme. Therefore, CPB was being turned over by PDC, in addition to inactivating it. CPB can be labeled a suicide-type inactivator for PDC.

Binding Sites↗
PubMed1982

Synthesis of 9-(3,4-dioxopentyl)hypoxanthine, the first arginine-directed purine derivative: an irreversible inactivator for purine nucleoside phosphorylase.

The synthesis of two potential arginine-directed purine-based analogues, 6-chloro-9-(3,4-dioxopentyl)purine (6) and 9-(3,4-dioxopentyl)hypoxanthine (7), is reported. Compound 7 was extensively tested as a potential affinity label of purine nucleoside phosphorylase (EC 2.4.2.1) from human erythrocytes. Evidence that 7 reacted with the catalytic center of purine nucleoside phosphorylase includes the following: (1) time-dependent inactivation of the enzyme by 7 was observed; (2) a plot of the pseudo-first-order rate constant for inactivation of the enzyme vs. concentration of 7 was hyperbolic, characteristic of saturation phenomenon; (3) substrates (Pi, arsenate, inosine) and a competitive inhibitor (formycin B) protected the enzyme from inactivation by 7. Compound 7 was 25 times more effective in inhibiting purine nucleoside phosphorylase than butanedione. Evidence that 7 modified arginine(s) includes the following: (1) when the inactivation was performed in borate, both the rate and the extent of inactivation were enhanced compared to those of the controls run in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer; (2) dialysis of inactivator reversed the inactivation in Tris-HCl but not in borate buffer. All the above evidence combined with the previous demonstration [Jordan, F., & Wu, A. (1978) Arch. Biochem. Biophys. 190, 699-704] that butanedione modified only arginines in purine nucleoside phosphorylases and the results presented here demonstrating the similarities in the behavior of butanedione and 7 imply that compound 7 can be called an arginine-directed affinity label for purine nucleoside phosphorylase.

Hypoxanthines↗
PubMed1981

Proton nuclear magnetic resonance evidence for the absence of a stable hydrogen bond between the active site aspartate and histidine residues of native subtilisins and for its presence in thiolsubtilisins.

The very low field proton nuclear magnetic resonance (1H NMR) found in aqueous solutions of serine proteases and their zymogens is characteristic of the hydrogen bond between the imidazolium and aspartate groups of the catalytic triad: Ser-His-Asp [Robillard, G., & Shulman, R. G. (1972) J. Mol. Biol. 71, 507--511]. According to 1H correlation NMR spectroscopic studies performed in 80/20 (v/v) H2O/2H2O, no such resonance is found in native subtilisins (even at -2 degrees C and pH 6.0), but it is present in thiolsubtilisins and in the phenylboronic acid derivatives of the serine enzymes. The resonance was not visible in the mercuric or carboxamidomethyl derivatives of the thiol enzymes or in the phenylboronic acid--serine enzyme complex if the serine enzyme was first acylated with phenylmethanesulfonyl fluoride. The histidine at the catalytic site of thiolsubtilisin carries a positive charge between pH 5.6 and 8.4, in accord with previous data in favor of a mercaptide--imidazolium ion pair at the catalytic site. The charge distribution (- + -) at the active site of thiolsubtilisin and in the phenylboronic acid derivatives of the serine enzymes resembles that in the tetrahedral transition state formed between a serine enzyme and its substrate. Therefore, the stable hydrogen bond (found in the thiol enzyme and in the phenylboronic acid derivative of the serine enzyme) should be more important during catalysis than in the substrate-free enzyme.

Aspartic Acid↗
PubMed1979

Purine nucleoside phosphorylase cleaves the C--O bond of ribose 1-phosphate. Evidence from the 18O shift in 31P NMR.

An equilibrium mixture of highly enriched [18(O)]Pi (represents the mixture of [[18(O)4]Pi, [[18(O)3]Pi, [18(O)2]Pi as represented in the figures, unless otherwise specified), alpha-D-ribose 1-[16(O)]phosphate, and hypoxanthine plus inosine was equilibrated with calf spleen purine-nucleoside phosphorylase (EC 2.4.2.1). The 31P NMR spectrum clearly indicated the formation of alpha-D-ribose 1-[18(O)4]-phosphate and of [16(O)]Pi. Incubation for the same time span in the absence of alpha-D-ribose 1-phosphate left the [18(O)4]Pi isotopic distribution unchanged. The results clearly demonstrated that the C--O bond of alpha-D-ribose 1-phosphate is cleaved in the enzymatic reaction. It is unlikely that the enzyme catalyzes the exchange of oxygen between Pi and H2O. Several possible mechanistic pathways are ruled out by the results, which demand attack by a phosphate oxygen at the anomeric C-1' atom.

Animals↗
PubMed1978

Stereoelectronic factors in the binding of substrate analogues and inhibitors to purine nucleoside phosphorylase isolated from human erythrocytes.

Several aspects of the stereoelectronic requirements of substrates of human erythrocytic purine nucleoside phosphorylase (E.C. 2.4.2.1) were elucidated providing the following information: (a) the N1 position cannot have a nonhydrogen substituent; (b) the 5'-OH group must be present for catalytic activity to be exhibited but is not an essential functional group for inhibitory action to be observed; (c) on the C8 position groups larger than -NH2 or -Br cannot be accommodated; (d) the syn-glycosyl conformation (i.e., 8-bromoguanosine) is acceptable but may not be an absolute requirement for phosphorolysis; (e) among nucleic base inhibitors methylation at N3, N7, or N9 vastly decreases the inhibitory properties as does a nitrogen in lieu of C-H in the 8 position. The results clearly indicate that this enzyme differs in its stereoelectronic requirements from the Escherichia coli enzyme.

Binding, Competitive↗
PubMed1977

C8-amino purine nucleosides. A well-defined steric determinant of glycosyl conformational preferences.

100 MHz proton magnetic resonance measurements were performed on dilute solutions of adenosine and guanosine and their 8MH2, 8-NHCH3 8-N(CH3)2 and 8-bromo derivatives. The chemical shift of the ribose C2'-H and especially the difference in chemical shifts between the C1'-H and C2'-H resonances clearly indicated whether the nucleoside exists in a syn glycosyl conformation (the C8-dimethylamino derivatives) or as a flexible syn-anti mixture (the monomethylamino and amino derivatives). The temperature dependent behavior of these indicators can be employed to define qualitative shifs in syn-anti equilibrium with temperature. An increased C1'-H-C2'-H chemical shift separation implies shift to more anti, a decreased separation a shift to more syn conformers.

Adenosine↗
PubMed1977

Solvent effects on thiamin-enzyme model interactions. I. Interactions with tryptophan.

The solvent polarity dependence of the interaction between thiamin and tryptophan was studied by spectrophotometric methods. The ultraviolet (UV) data clearly indicate that the interaction is weakened when the complex is transferred from water to aqueous ethanol or aqueous dioxane. The interaction of thiamin and tryptophan could also be detected by fluorescence-quenching studies (excitation of tryptophan at 287 nm, maximum emission at 348 nm). Appropriate treatment of the quenching data allowed dissection into static and dynamic contributions. A pyrimidine derivative related to thiamin, both in its neutral and protonated states, was shown to interact with tryptophan by fluorescence techniques, but not by UV. A thiazolium model was shown to interact with tryptophan by UV but was an inefficient quencher of the tryptophan fluorescence. Theoretical models are presented to explain the solvent dielectric constant dependence of the association constant between tryptophan and thiamin. Both electrostatic and dispersion forces are found to contribute to the stability of the complex.

Binding Sites↗