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B Zerner

Publications and source records attributed to B Zerner.

At least 19 recordsLinked to original sources
PubMed1996

New catalytic roles for serine esterases: a 19F-NMR study of the interaction of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone with chicken liver carboxylesterase.

The reactions of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone (TDPP) with chicken liver carboxylesterase have shown that this ketone hydrate is not only a potent inhibitor of the enzyme, but also a substrate for a number of enzyme-catalyzed reactions. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is initially dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent ketone then produces a hemiketal adduct. However, the slow reactivation (by dialysis) of TDPP-inhibited enzyme indicates that the interaction with this inhibitor is more complex. At equilibrium, a dissociation constant of 2.4 pM was obtained for this interaction. 19F-NMR studies of the enzyme-TDPP complex show that after pre-equilibration, the major adduct is not the hemiketal adduct. It is proposed that this final adduct is a cross-linked adduct formed between TDPP, the active-site serine and the active-site histidine. 19F-NMR studies reveal that chicken liver carboxylesterase catalyses the cleavage of TDPP to yield either fluoride ion or trifluoroacetate, and also the benzilic acid rearrangement of TDPP to alpha-trifluoromethylmandelate. These products have also been identified in model studies of the reaction between TDPP and imidazole.

Acetone↗
PubMed1996

Inhibition of chicken liver carboxylesterase by activated carbonyls and carbonyl hydrates.

Identical Kcat values (approximately 40 s-1) are obtained for the chicken liver carboxylesterase catalyzed hydrolysis of phenyl, p-nitrophenyl and o-nitrophenyl benzoates providing support for the involvement of an acyl-enzyme pathway, with the rate-limiting deacylation of a common benzoyl-enzyme intermediate. Chicken liver carboxylesterase catalyzed fragmentation of (E)-benzilmonoxime O-2,4-dinitrophenyl ether shows a pH dependence on a group active in the free base form with a pK'a approximately 5.0. The Ki-pH profile for benzil inhibition shows a dependence on a similar group with a pK'a = 5.4. The reactions between chicken liver carboxylesterase and the hydrated aldehyde, chloral hydrate, have shown this compound to be at once a substrate and potent inhibitor of the enzyme. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is first dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent aldehyde produces a hemiacetal adduct. The Ki value for chloral hydrate inhibition calculated from the kinetic analysis (90 nM) compares favourably with the value measured from the steady-state kinetics (87 nM).

Animals↗
PubMed1991

Cloning and sequencing of a jack bean urease-encoding cDNA.

A cDNA which encodes the entire amino acid (aa) sequence of the mature jack bean urease has been cloned in Escherichia coli from a library prepared from the mRNA of developing jack beans. It was necessary to use reverse transcriptase in the cDNA was obtained in the form of two contiguous DNA fragments, each of which was completely sequenced. The conceptual translation of the nt sequence gave an 840-aa sequence which was identical to the directly determined sequence except for one conservative aa substitution (Takashima et al., Eur. J. Biochem. 175 (1988) 151-165). These data constitute the first report on the cloning and sequence of the cDNA encoding a urease from any higher plant.

Amino Acid Sequence↗
PubMed1985

The relationship between the carboxylesterase and monoacylglycerol lipase activities of chicken liver microsomes.

The carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) and monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) activities, measured against ethyl butyrate and emulsified monooleoylglycerol respectively, were determined for chicken liver microsomes and highly purified chicken liver carboxylesterase. The activity ratio (ethyl butyrate activity/monooleoylglycerol activity) was approx. 5 for microsomes and approx. 400 for carboxylesterase. Homogenization of microsomes in 0.1 M Tris-HCl buffer (pH 7.92) released all of the ethyl butyrate activity and about half of the monooleoylglycerol activity into a soluble form. Both activities eluted from a Sephadex G-200 column with the same elution volume as that of pure carboxylesterase. This fraction (fraction B) had an activity ratio of approx. 15, an average pI of 5.01 (cf. 4.75 for carboxylesterase), and ran on polyacrylamide gel electrophoresis at pH 8.6 as a number of closely spaced esterase bands with mobilities considerably less than those of the esterase bands present in the carboxylesterase. Fraction B activities against both substrates were completely inhibited by diethyl p-nitrophenyl phosphate and completely precipitated by antibody to carboxylesterase. The remaining half of the monoacylglycerol lipase activity of microsomes was solubilized by treatment with 1.5% (w/v) Triton X-100. This solubilized monoacylglycerol lipase was completely inhibited by diethyl p-nitrophenyl phosphate, showing it to be a serine-dependent enzyme like the carboxylesterases. However, it had no detectable activity against ethyl butyrate, indicating that it is not closely related to the carboxylesterases.

Animals↗
PubMed1984

Enzymatically active zinc, copper and mercury derivatives of the one-iron form of pig allantoic fluid acid phosphatase.

Derivatives of the violet, iron-containing acid phosphatase of pig allantoic fluid have been prepared in which one of the two iron atoms present in the native enzyme has been replaced by zinc, copper or mercury. The derivatives so formed are enzymatically active: the Zn-Fe, Cu-Fe and Hg-Fe enzymes have specific activities of about 80%, 25% and 17% respectively, of the maximum specific activity of the Fe-Fe enzyme in the standard assay at pH 4.9 with p-nitrophenyl phosphate as substrate. In contrast to the Fe-Fe enzyme, the mixed metal derivatives are not rapidly inactivated by H2O2. Visible absorption spectra of the derivatives confirm that all of the visible absorption of the Fe-Fe enzyme is due to one of the iron atoms. Attempts to prepare an active Cu-Cu enzyme were unsuccessful.

Acid Phosphatase↗
PubMed1984

Urea and urease.

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Animals↗
PubMed1983

Mössbauer and EPR study of the binuclear iron centre in purple acid phosphatase.

Mössbauer spectra have been determined on 57Fe-enriched samples of both pink (reduced) and purple (oxidized) forms of pig allantoic acid phosphatase (EC 3.1.3.2), and EPR spectra on corresponding unenriched samples. The spectra show unambiguously that both forms of the enzyme contain two distinct, antiferromagnetically coupled, high-spin iron atoms: a ferrous-ferric ion pair in the pink, reduced form, and a pair of ferric ions in the purple, oxidized form.

Acid Phosphatase↗
PubMed1980

Jack bean urease (EC 3.5.1.5). I. A simple dry ashing procedure for the microdetermination of trace metals in proteins. The nickel content of urease.

A simple and inexpensive procedure for determination of microgram quantities of metal ions in proteins is described and tested with nickel and iron. The method involves (a) dry ashing in an oxygen atmosphere at 450-460 degrees C in Pyrex vessels, (b) conversion of the metal oxides or other compounds to readily soluble species, and (c) spectrophotometric analysis. An improved procedure for the direct spectrophotometric determination of nickel using dimethylglyoxime is accurate to +/- 2% or better with samples of 1-5 microgram of nickel. These techniques were used to determine that the nickel content of freshly prepared jack bean urease in 2.00 +/- 0.12 g-at./96 600 g protein. The corresponds to 2.0 nickel ions per subunit. This result was confirmed by atomic absorption analysis, which also showed that calcium, manganese, cobalt, and iron are not present in significant amounts in urease.

Diacetyl↗
PubMed1980

Jack been urease (EC 3.5.1.5). II. The relationship between nickel, enzymatic activity, and the "abnormal" ultraviolet spectrum. The nickel content of jack beans.

At low pH, EDTA promotes the loss of the tightly bound nickel ions from jack bean urease. The specific activity of soluble enzyme after partial EDTA-promoted inactivation is a linear function of the nickel content. The results are consistent with the presence of 2.0 nickel ions per 97 000-dalton subunit in pure urease. The time scale for loss of enzymatic activity and nickel under these conditions is similar to that for loss of the "abnormal" tail absorption in the ultraviolet and visible absorption spectrum of urease (including the shoulder at approximately 420 nm). This indicates that nickel in urease is essential for enzymatic activity and establishes that the metal ions are in part responsible for the tail absorption in the ultraviolet spectrum of urease. After partial inactivation in the presence of EDTA either at low pH or in 2.5 M guanidinium chloride at neutral pH, urease did not regain activity in the presence of Ni2+. As yet apourease has not been produced reversibly. Jack bean seeds grown hydroponically without added nickel were low in both urease activity and nickel (10 and 6%, respectively, of parent seeds). Several other metal ions were readily available. This result suggests that metal ions other than nickel cannot substitute for nickel in the formation of normally active urease.

Diacetyl↗
PubMed1980

Jack bean urease (EC 3.5.1.5). III. The involvement of active-site nickel ion in inhibition by beta-mercaptoethanol, phosphoramidate, and fluoride.

Interaction of beta-mercaptoethanol with urease produces large, rapid and fully reversible spectral changes in that part of the electronic absorption spectrum which is associated with the tightly bound nickel ions. The spectrophotometrically determined value of the dissociation constant of the beta-mercaptoethanol-urease complex (0.9 +/- 0.05 mM at pH 7.12 and 25 degrees C) is in agreement with the Ki (0.72 +/- 0.26 mM) for beta-mercaptoethanol acting as a competitive inhibitor in the hydrolysis of urea. This constitutes direct evidence that the nickel in jack bean urease is at the active site. Inhibition of urease by phosphoramidate is slowly achieved and slowly reversed, and upon reactivation of the isolated phosphoramidate-urease complex, phosphoramidate is regenerated in good yield. Spectrophotometric experiments indicate that phosphoramidate binds to nickel ion in urease. Competition with beta-mercaptoethanol was used to determine a dissociation constant (1.23 +/- 0.10 mM at pH 7.12 and 25 degrees C) for a fluoride-evidence is presented which indicates that in the presence of urea, a ternary complex (fluoride-urea-urease) is formed.

Amides↗
PubMed1980

Jack bean urease (EC 3.5.1.5). IV. The molecular size and the mechanism of inhibition by hydroxamic acids. Spectrophotometric titration of enzymes with reversible inhibitors.

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 +/- 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of beta-mercaptoethanol is approximately 95 000. Essentially the same subunit molecular weight (approximately 93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride - beta-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein-inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.

Amino Acids↗