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H C Rilling

Publications and source records attributed to H C Rilling.

At least 37 records · Page 2Linked to original sources
PubMed1981

Crystallization and partial characterization of dimethylallyl pyrophosphate: L-tryptophan dimethylallyltransferase from Claviceps sp. SD58.

Dimethylallyl pyrophosphate: L-tryptophan dimethylallyltransferase (dimethylallyl tryptophan synthetase) has been purified from Claviceps strain SD58 to a homogeneous crystalline form. The enzyme is pure as judged by polyacrylamide gel electrophoresis and contains two similar subunits of 34,000 molecular weight as shown by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Since the enzyme was determined to have a molecular weight of 70,000 by gel exclusion chromatography, it is dimeric. The substrates dimethylallyl pyrophosphate and tryptophan each have mixed (negative to positive) cooperativity with a minimum Hill coefficient of 0.37 and 0.73, respectively. Calcium ion is a positive allosteric effector and, at 20 mM concentration, deregulates the enzyme. The deregulated enzyme has Km values of 7.2 microM for dimethylallyl pyrophosphate and 8.8 microM tryptophan. The Vmax under deregulated conditions is 953 nmol/min/mg, giving a turnover number of 14. Ca2+ has a S[0.5] of 4.5 mM. The kinetic mechanism is random sequential.

Alkyl and Aryl Transferases↗
PubMed1981

Isolation and characterization of a photoaffinity-labeled peptide from the catalytic site of prenyltransferase.

Previously we presented evidence for the selective modification of the catalytic site of prenyltransferase by photoaffinity labeling with o-azidophenylethyl pyrophosphate [Brems, D. N., & Rilling, H. C. (1979) Biochemistry 18, 860]. In the present work, we report the isolation and characterization of a CNBr fragment of 30 amino acid residues from the photoaffinity-labeled enzyme. This CNBr fragment contains over 809% of the total label attached to prenyltransferase as a result of photoaffinity labeling. Several lines of evidence indicate that a number of residues in this CNBr fragment have been modified. First, Edman degradation of this labeled peptide demonstrates that at least 16 of the 30 amino acids have been modified by the photoaffinity reagent. The two most extensively modified amino acids are a specific arginine and alanine. Second, two-dimensional chromatography of Pronase digestions of the labeled CNBr fragment indicates that at least 11 different products resulted from photoaffinity labeling. Third, peptide maps of a trypsin digest of this CNBr fragment show that the attached affinity label is distributed among at least three of the resulting products of tryptic hydrolysis. Finally, comparison of amino acid analysis of this CNBr fragment with that of its counterpart isolated from native enzyme is consistent with the modification of a number of amino acids rather than a few y the photoaffinity labeling process.

Affinity Labels↗
PubMed1979

Photoaffinity labeling of the catalytic site of prenyltransferase.

Three photoreactive substrate analogues, o-azidophenethyl pyrophosphate, p-azidophenethyl pyrophosphate, and 3-azido-1-butyl pyrophosphate, have been synthesized as site-directed probes to label the catalytic site of prenyltransferase. Due to the relatively poor affinity of p-azidophenethyl pyrophosphate and 3-azido-1-butyl pyrophosphate for the enzyme, only o-azidophenethyl pyrophosphate (aryl azide) was utilized for photoaffinity labeling. This aryl azide has a UV absorption maximum at 250 nm. In the absence of activating light, binding studies demonstrate that the o-aryl azide competes for binding with both the natural substrates, isopentenyl pyrophosphate and geranyl pyrophosphate. More than 90% enzymatic activity is lost when enzyme is irradiated in the presence of the aryl azide as compared to irradiation in the absence of the azide, and the protein loses its capacity for substrate binding in direct proportion to photolabeling. A stoichiometry of 2 mol of affinity label covalently bound per mol of enzyme dimer was established with [1-3H]-o-azidophenethyl pyrophosphate. Since there are two catalytic sites per enzyme dimer, the o-aryl azide appears specifically to label the enzyme at its catalytic sites. Additional evidence that the reagent was specific for the catalytic site came from the observation that farnesyl pyrophosphate afforded complete protection against photoinactivation, while isopentenyl pyrophosphate provided partial protection. Gel isoelectric focusing verified this stoichiometry and indicated that the labeled enzyme has a more acidic isoelectric point than the native enzyme.

Affinity Labels↗
PubMed1978

Polyisoprenoid amphiphilic compounds as inhibitors of squalene synthesis and other microsomal enzymes.

Analogues of farnesyl pyrophosphate containing a farnesyl moiety and a variety of amine residues replacing the pyrophosphate have been synthesized. Most of these compounds were effective inhibitors of the synthesis of squalene and presqualene pyrophosphate from farnesyl pyrophosphate. 50% inhibition was obtained at concentrations between 50 and 100 micron. These analogues also inhibited other microsomal enzymes so they apparently function as general inhibitors of microsomal enzymes.

Cytochrome Reductases↗
PubMed1976

Substrate Binding of avian liver prenyltransferase.

Prenyltransferase (farnesyl pyrophosphate synthetase) was purified from avian liver and characterized by Sephadex and sodium dodecyl sulfate gel chromatography, peptide mapping, and end-group analysis. The enzyme is 85 800 +/- 4280 daltons and consists of two identical subunits as judged by sodium dodecyl sulfate gel electrophoresis, peptide mapping, and end-group analysis. Chemical analysis of the protein revealed no lipid or carbohydrate components. Avian prenyltransferase synthesizes farnesyl pyrophosphate from either dimethylallyl or geranyl pyrophosphate and isopentenyl pyrophosphate. A lower rate of geranylgeranyl pyrophosphate synthesis from farnesyl pyrophosphate and isopentenyl pyrophosphate was also demonstrated. Michaelis constants for farnesyl pyrophosphate synthesis are 0.5 muM for both isopentenyl pyrophosphate and geranyl pyrophosphate. The V max for the reaction is 1990 nmol min-1 mg-1 (170 mol min-1 mol-1 enzyme). Substrate inhibition by isopentenyl pyrophosphate is evident at high isopentenyl pyrophosphate and low geranyl pyrophosphate concentrations. Michaelis constants for geranylgeranyl pyrophosphate synthesis are 9 muM for farnesyl pyrophosphate and 20 muM for isopentenyl pyrophosphate. The Vmax is 16 nmol min-1 mg-1 (1.4 mol min-1 mol-1 enzyme). Two moles of each of the allylic substrates is bound per mol of enzyme. The apparent dissociation constants for dimethylallyl, geranyl, and farnesyl pyrophosphates are 1.8, 0.17, and 0.73 muM, respectively. Dimethylallyl and geranyl pyrophosphates bound competitively to prenyltransferase with one-for-one displacement. Four moles of isopentenyl pyrophosphate was bound per mole of enzyme. Citronellyl pyrophosphate, an analogue of geranyl pyrophosphate, was competitive with the binding of 2 of the 4 mol of isopentenyl pyrophosphate bound. The data are interpreted to indicate that each subunit of avian liver prenyltransferase has a single allylic binding site accommodating dimethylallyl, geranyl, and farnesyl pyrophosphates, and one binding site for isopentenyl pyrophosphate. In the absence of an allylic pyrophosphate or analogue, isopentenyl pyrophosphate also can bind to the allylic site.

Alkenes↗
PubMed1976

Prenyltransferase: the mechanism of the reaction.

The enzyme, prenyltransferase, which normally catalyzes the addition of an allylic pyrophosphate to isopentenyl pyrophosphate, has been found to catalyze the hydrolysis of its allylic substrate. The rate of this hydrolysis is markedly stimulated by inorganic pyrophosphate. Competition experiments with 2-fluoroisopentenyl pyrophosphate and inorganic pyrophosphate demonstrated that inorganic pyrophosphate stimulated hydrolysis by binding at the isopentenyl pyrophosphate site. Hydrolysis carried out in H218O or with (1S)-[1-3H]geranyl pyrophosphate show the C-O bond is broken and the C1 carbon of geranyl pyrophosphate is inverted in the process. These results are interpreted to favor a carbonium ion mechanism for the prenyltransferase reaction.

Animals↗
PubMed1975

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.

Alkenes↗
PubMed1975

Crystallization and partial characterization of prenyltransferase from avian liver.

Prenyltransferase (EC 2.5.1.1) has been obtained from chicken liver in a stable crystalline form. The enzyme has been shown to be homogeneous by polyacrylamide gel electrophoresis at pH 8.4, and by electrophoresis in sodium dodecyl sulfate containing gels. Electrofocusing of the crystalline enzyme results in a single sharp protein peak with a pI of 5.72. The protein is a dimer of molecular weight 86,000 whose subunits were not resolved by gel electrophoresis in sodium dodecyl sulfate. Michaelis constants of 0.5 muM for both isopentenyl pyrophosphate and geranyl pyrophosphate are 3-20-fold lower than those found for prenyltransferase from yeast or pig liver (Eberhardt, N., and Rilling, H. C. (1974), J. Biol. Chem. (in press); Dorsey, J. K., Dorsey, J. A., and Porter, J. W. (1966), J. Biol. Chem. 241, 5353; Holloway, P. W., and Popjak, G. (1967), Biochem. J. 104, 57). The enzyme primarily synthesizes farnesyl pyrophosphosphate from dimethylallyl or geranyl pyrophosphate although some geranylgeranyl pyrophosphate is formed under certain conditons. This is the first preparation of a stable crystalline enzyme of sterol and terpene biosynthesis.

Alkenes↗