Biomedical subjects

M A Ator

Publications and source records attributed to M A Ator.

26 records · Page 2Linked to original sources

PubMed1989

Mechanism and inhibition of delta 24-sterol methyltransferase from Candida albicans and Candida tropicalis.

The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed.

Candida↗

PubMed1989

Stabilized isoporphyrin intermediates in the inactivation of horseradish peroxidase by alkylhydrazines.

The reaction of horseradish peroxidase with alkylhydrazines results in delta-meso-alkylation of the prosthetic heme group and enzyme inactivation (Ator, M. A., David, S. K., and Ortiz de Montellano, P. R. (1987) J. Biol. Chem. 262, 14954-14960). As reported here, enzyme inactivation is associated with the accumulation of intermediates that absorb at approximately 835 nm. The properties of these intermediates, including their collapse to give meso-alkylhemes, identify them as isoporphyrins. The t1/2 values for inactivation and formation of the isoporphyrin intermediate at 25 degrees C are, respectively, 11.6 and 12.5 min for methylhydrazine (2.0 mM), 8.7 and 7.2 min for ethylhydrazine (1.0 mM), and 30 and 25 s for phenylethylhydrazine (50 microM). The isoporphyrin intermediates are surprisingly long-lived, with half-lives (35 degrees C, pH 7.0) of 9, 28, 96, and 450 min for, respectively, the phenylethyl, methyl, n-butyl, and ethyl analogues. pH studies show that protonation of a group with pKa = 5.0-6.5 accelerates isoporphyrin decay and decreases steady state isoporphyrin accumulation. Horseradish peroxidase reconstituted with delta-meso-methylheme, unlike horseradish peroxidase with a heme that has a larger meso-substituent, is catalytically active but is more sensitive to H2O2-mediated degradation of the prosthetic group than is the native enzyme. The delta-meso-methylheme prosthetic group is converted in the reaction with H2O2 to a biliverdin-like product. The results implicate highly stabilized isoporphyrin intermediates in the inactivation of horseradish peroxidase by alkylhydrazines and indicate that inactivation by the meso-alkyl groups is due to steric interference with electron delivery to the heme edge rather than to intrinsic electronic consequences of meso-alkylation. The structural features that stabilize the cationic isoporphyrins may also be involved in stabilization of the Compound I porphyrin radical cation.

Alkylation↗

PubMed1988

Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX.

Catalytic turnover of sodium azide by horseradish peroxidase, which produces the azidyl radical, results in inactivation of the enzyme with KI = 1.47 mM and kinact = 0.69 min-1. Inactivation of 80% of the enzyme requires approximately 60 equiv each of NaN3 and H2O2. The enzyme is completely inactivated by higher concentrations of these two agents. meso-Azidoheme as well as some residual heme are obtained when the prosthetic group of the partially inactivated enzyme is isolated and characterized. Reconstitution of horseradish peroxidase with meso-azidoheme yields an enzyme without detectable catalytic activity even though reconstitution with heme itself gives fully active enzyme. The finding that catalytically generated nitrogen radicals add to the meso carbon of heme shows that biological meso additions are not restricted to carbon radicals. The analogous addition of oxygen radicals may trigger the normal and/or pathological degradation of heme.

Azides↗

PubMed1987

Structure and catalytic mechanism of horseradish peroxidase. Regiospecific meso alkylation of the prosthetic heme group by alkylhydrazines.

Horseradish peroxidase is inactivated in a time-, H2O2-, and concentration-dependent manner by phenylethyl-, ethyl-, and methylhydrazine. The pseudo- first order kinetic constants for these inactivation reactions at pH 7 are: phenylethyl (KI = 115 microM, kinact = 1.5 min-1, partition ratio = 11), ethyl (KI = 145 microM, kinact = 0.08 min-1, partition ratio = 32), and methyl (KI = 3000 microM, kinact = 0.12 min-1, partition ratio = 80). At pH 5, the constants for the phenylethyl reaction change to KI = 1540 microM and kinact = 0.86 min-1. A transient absorbance at approximately 830 nm, suggestive of an isoporphyrin intermediate, is seen during these reactions. The prosthetic heme is converted by each of the three alkylhydrazines into the corresponding delta-meso-alkylated heme. Complete inactivation of the enzymes by methyl-, ethyl-, and phenylethylhydrazine is associated with alkylation of 60-70, 70, and 90%, respectively, of the prosthetic heme groups. The absence of N-alkylation and the high specificity for the delta-meso position, even with agents as small as methylhydrazine, strengthen the proposal that electron abstraction is mediated by the heme edge rather than the ferryl oxygen of horseradish peroxidase.

Alkylation↗

PubMed1987

Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate.

Ribonucleoside diphosphate reductase (RDPR) from Escherichia coli was completely inactivated by 1 equiv of the mechanism-based inhibitor 2'-azido-2'-deoxyuridine 5'-diphosphate (N3UDP). Incubation of RDPR with [3'-3H]N3UDP resulted in 0.2 mol of 3H released to solvent per mole of enzyme inactivated, indicating that cleavage of the 3' carbon-hydrogen bond occurred in the reaction. Incubation of RDPR with [beta-32P]N3UDP resulted in stoichiometric production of inorganic pyrophosphate. One equivalent of uracil was eliminated from N3UDP, but no azide release was detected. Analysis of the reaction of RDPR with [15N3]N3UDP by mass spectrometry revealed that the azide moiety was converted to 0.9 mol of nitrogen gas per mole of enzyme inactivated. The tyrosyl radical of the B2 subunit was destroyed during the inactivation by N3UDP as reported previously [Sjöberg, B.-M., Gräslund, A., & Eckstein, F. (1983) J. Biol. Chem. 258, 8060-8067], while the specific activity of the B1 subunit was reduced by half. Incubation of [5'-3H]N3UDP with RDPR resulted in stoichiometric covalent radiolabeling of the enzyme. Separation of the enzyme's subunits by chromatofocusing revealed that the modification was specific for the B1 subunit.

Affinity Labels↗

PubMed1987

Protein control of prosthetic heme reactivity. Reaction of substrates with the heme edge of horseradish peroxidase.

Incubation of horseradish peroxidase with phenylhydrazine and H2O2 markedly depresses the catalytic activity and the intensity, but not position, of the Soret band. Approximately 11-13 mol of phenylhydrazine and 25 mol of H2O2 are required per mol of enzyme to minimize the chromophore intensity. The enzyme retains some activity after such treatment, but this activity is eliminated if the enzyme is isolated and reincubated with phenylhydrazine. The prosthetic heme of the enzyme does not react with phenylhydrazine to give a sigma-bonded phenyl-iron complex, as it does in other hemoproteins, but is converted instead to the delta-mesophenyl and 8-hydroxymethyl derivatives. The loss of activity is due more to protein than heme modification, however. The inactivated enzyme reacts with H2O2 to give a spectroscopically detectable Compound I. The results imply that substrates interact with the heme edge rather than with the activated oxygen of Compounds I and II and specifically identify the region around the delta-meso-carbon and 8-methyl group as the exposed sector of the heme. Horseradish peroxidase, in contrast to cytochrome P-450, generally does not catalyze oxygen-transfer reactions. The present results indicate that oxygen-transfer reactions do not occur because the activated oxygen and the substrate are physically separated by a protein-imposed barrier in horseradish peroxidase.

Chemical Phenomena↗

PubMed1986

Mechanism of ribonucleotide reductase from herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs.

Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of [3'-3H]ADP, [3'-H]UDP, and [5-3H] UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of [3'-3H]ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of [beta-32P]ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase (Harris, G., Ator, M., and Stubbe, J. A. (1984) Biochemistry 23, 5214-5225; Ator, M., and Stubbe, J. A. (1985) Biochemistry 24, 7214-7221).

Adenosine Diphosphate↗

PubMed1985

Mechanism of inactivation of Escherichia coli ribonucleotide reductase by 2'-chloro-2'-deoxyuridine 5'-diphosphate: evidence for generation of a 2'-deoxy-3'-ketonucleotide via a net 1,2 hydrogen shift.

Sodium borohydride or ethanethiol protects the Escherichia coli ribonucleoside-diphosphate reductase (RDPR) from inactivation by 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP). Incubation of [3'-3H]ClUDP with RDPR in the presence of NaBH4 allowed trapping of [3H]-2'-deoxy-3'-ketouridine 5'-diphosphate. Degradation of the reduced ketone by a combination of enzymatic and chemical methods indicated that the hydrogen originally present in the 3'-position of ClUDP is transferred to the beta-face of the 2'-position of 2'-deoxy-3'-keto-UDP. RDPR therefore catalyzes a net 1,2 hydrogen shift. Incubation of RDPR with ClUDP in the presence of ethanethiol allowed trapping of 2-methylene-3(2H)-furanone, the species responsible for inactivation of RDPR. Trapped 2-[(ethylthio)methyl]-3(2H)-furanone was identical by 1H NMR spectroscopy with material synthesized chemically. Both subunits of the enzyme are covalently radiolabeled in the reaction of RDPR with [5'-3H]ClUDP. Studies with [3'-3H]ClUDP and prereduced RDPR in the absence of a reductant and with oxidized RDPR indicated that the redox-active thiols of the B1 subunit are not involved in inactivation of the enzyme by ClUDP.

Borohydrides↗