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D W Pettigrew

Publications and source records attributed to D W Pettigrew.

At least 37 records · Page 2Linked to original sources
PubMed1987

Inactivation of Escherichia coli glycerol kinase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine: protection by the hydrolyzed reagent.

Incubation of Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSO2BzAdo) at pH 8.0 and 25 degrees C results in the loss of enzyme activity, which is not restored by the addition of beta-mercaptoethanol or dithiothreitol. The FSO2BzAdo concentration dependence of the inactivation kinetics is described by a mechanism that includes the equilibrium binding of the reagent to the enzyme prior to a first-order inactivation reaction in addition to effects of reagent hydrolysis. The hydrolysis of the reagent has two effects on the observed kinetics. The first effect is deviation from pseudo-first-order kinetic behavior due to depletion of the reagent. The second effect is the novel protection of the enzyme from inactivation due to binding of the sulfonate hydrolysis product. The rate constant for the hydrolysis reaction, determined independently from the kinetics of F- release, is 0.021 min-1 under these conditions. Determinations of the reaction stoichiometry with 3H-labeled FSO2BzAdo show that the inactivation is associated with the covalent incorporation of 1.08 mol of reagent/mol of enzyme subunit. Ligand protection experiments show that ATP, AMP, dAMP, NADH, 5'-adenylyl imidodiphosphate, and the sulfonate hydrolysis product of FSO2BzAdo provide protection from inactivation. The protection obtained with ATP is not dependent on Mg2+. Less protection is obtained with glycerol, GMP, etheno-AMP, and cAMP. No protection is obtained with CMP, UMP, TMP, etheno-CMP, GTP, or fructose 1,6-bisphosphate. The results are consistent with modification by FSO2BzAdo of a single adenine nucleotide binding site per enzyme subunit.

Adenosine↗
PubMed1987

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of the steady-state kinetic and proton relaxation rate properties of the Mn(II)-containing enzyme.

The essential Zn(II) in bovine liver dihydropyrimidine amidohydrolase (DHPase) was removed by incubation with 2,6-dipicolinic acid and replaced with Mn(II). Electron paramagnetic resonance studies of Mn(II) binding show that there are four binding sites per tetramer, and the dissociation constant at pH 7.5 is 13.5 microM. The substitution of Mn(II) for Zn(II) increases the specific activity of the enzyme approximately sixfold but has only a small effect (twofold increase) on the Km for 5-bromo-5,6-dihydrouracil (BrH2Ura). The pH dependence of the catalytic properties of Mn(II)-DHPase is the same as for the Zn(II) enzyme (Lee, M., Cowling, R., Sander, E., and Pettigrew, D. (1986) Arch. Biochem. Biophys. 248, 368-378). The pH dependence is well described in terms of the ionization of a single group with a pK of about 6 in the free enzyme. The ionization of this group is required for catalytic activity. The substitution of Mn(II) for Zn(II) does not affect the pH dependence of DHPase catalysis and therefore strongly suggests that the ionizable group is an amino acid residue at or near the active site, rather than a metal-bound water molecule. The pH dependence of the enhancement of the paramagnetic effect of the DHPase-Mn complex on the relaxation rate of the solvent water protons also is well described in terms of the ionization of a single group with a pK of about 6. Ionization of the group which is involved in catalysis also perturbs the environment of the bound Mn(II). The ionization of the active site group does not affect the number of exchangeable water molecules but does affect the symmetry of the environment of the bound Mn(II) and its electron relaxation.

Algorithms↗
PubMed1986

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites.

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1986

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of inactivation by chelators and steady-state kinetic properties.

Dihydropyrimidine amidohydrolase (EC 3.5.2.2) catalyzes the reversible hydrolysis of 5,6-dihydropyrimidines to the corresponding beta-ureido acids. Previous work has shown that incubation of this Zn2+ metalloenzyme with 2,6-dipicolinic acid, 8-hydroxyquinoline-5-sulfonic acid, or o-phenanthroline results in inactivation by Zn2+ removal by a reaction pathway involving formation of a ternary enzyme-Zn2+-chelator complex which subsequently dissociates to yield apoenzyme and the Zn2+-chelate (K. P. Brooks, E. A. Jones, B. D. Kim, and E. G. Sander, (1983) Arch. Biochem. Biophys. 226, 469-483). In the present work, the pH dependence of chelator inactivation is studied. The equilibrium constant for formation of the ternary complex is strongly pH dependent and increases with decreasing pH for all three chelators. There is a positive correlation between the value of the equilibrium constant observed for each chelator and the value of its stability constant for formation of Zn2+-chelate. The affinity of the chelators for the enzyme increases in the order 8-hydroxyquinoline-5-sulfonic acid greater than o-phenanthroline greater than 2,6-dipicolinic acid. The first-order rate constant for breakdown of the ternary complex to yield apoenzyme and Zn2+-chelate is invariant with pH for a given chelator but is different for each chelator, increasing in the reverse order. The pH dependence of the inactivation shows that two ionizable groups on the enzyme are involved in the inactivation. On the other hand, the steady-state kinetic behavior of the enzyme is well-described by ionization of a single group with a pK of 6.0 in the free enzyme. The basic form of the group is required for catalysis; protonation of the group decreases both Vmax and the apparent affinity for substrate. Conversely, binding of substrate decreases the pK of this group to about 5. L-Dihydroorotic acid is shown to be a competitive inhibitor of dihydropyrimidine amidohydrolase. Binding of L-dihydroorotic acid increases the pK of the ionizable group to 6.5. The agreement between the pK in the enzyme-L-dihydroorotic acid complex and the higher pK observed in the pH dependence of inactivation by chelators suggests that the same group is involved in the binding of acid, and chelators. The different effects of substrate and L-dihydroorotic acid on the pK suggest that the binding modes of these two ligands may be different and suggest a structural basis for the mutally exclusive substrate specificities of dihydropyrimidine amidohydrolase and dihydroorotase.

Amidohydrolases↗
PubMed1985

Evidence for a pH-dependent isomerization of Clostridium oroticum dihydroorotase.

Analytical gel permeation chromatography on both Sephadex and polyacrylamide columns shows that Clostridium oroticum dihydroorotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) undergoes a large decrease in molecular size when the pH is decreased from 8 to 6. The Stokes radius decreases from about 40 A to 36 A. Neither the molecular size nor kinetic properties are dependent on protein concentration. Thus, the decreased molecular size reflects a pH dependent isomerization of the enzyme.

Amidohydrolases↗
PubMed1985

Dihydro-orotase from Clostridium oroticum. Purification and reversible removal of essential zinc.

A new purification procedure involving five column-chromatography steps is described for dihydro-orotase (L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3) from Clostridium oroticum (A.T.C.C. 25750). The native purified enzyme is a dimer of Mr 102 000 and contains 4.0 +/- 0.3 g-atoms of zinc/mol of dimer. These observations agree with those reported previously [Taylor, Taylor, Balch & Gilchrist (1976) J. Bacteriol. 127, 863-873]. It is conclusively demonstrated that dihydro-orotase is a zinc metalloenzyme. Zinc is reversibly removed by treatment with chelators in phosphate buffer at pH 6.5, as demonstrated by atomic absorption spectrophotometry and decrease of enzyme activity. The specific activity is linearly dependent on zinc content. Addition of ZnSO4 to the chelator-treated enzyme results in regain of the normal complement of zinc and enzyme activity. Kinetic properties of the reconstituted enzyme are indistinguishable from those of the native enzyme. The amino acid composition of the homogeneous enzyme suggests that the zinc atoms occupy different environments.

Amidohydrolases↗
PubMed1985

Enzyme elements involved in the interconversion of L-carbamylaspartate and L-dihydroorotate by dihydroorotase from Clostridium oroticum.

Enzyme elements that are involved in the reversible cyclization of L-carbamylaspartate to L-dihdroorotate catalyzed by dihydroorotase (EC 3.5.2.3) from Clostridium oroticum (ATCC 25750) have been studied. Removal of Zn(II) from the enzyme by chelators followed by incubation of apoenzyme with Co(II) results in replacement of two to three of the four Zn(II) ions per molecule by Co(II). The catalytic properties of the Zn(II)Co(II) dihydroorotase are different from those of native enzyme. The Vmax is increased for both the synthesis and hydrolysis of L-dihydroorotate. The Km for L-dihydroorotate is unchanged, while the Km for L-carbamylaspartate is increased more than twofold. On the other hand, the kinetic properties of Zn(II)-reconstituted dihydroorotase are indistinguishable from those of native enzyme. The pH dependence of Vmax is also altered by the Co(II) substitution. For both Zn(II)- and Zn(II)Co(II)-dihydroorotase, this pH dependence is well described by a single ionization and the pK's for L-dihydroorotate synthesis and hydrolysis are different. Substitution with Co(II) increases the pK for both reaction directions to different extents. These results strongly support a role for the tightly bound metals in the catalytic mechanism. In addition, diethylpyrocarbonate rapidly inactivates the enzyme. The inactivation is prevented by L-dihydroorotate. This result is consistent with a role for at least one histidine in catalysis. The possibility that C. oroticum dihydroorotase may be useful model for the more complex mammalian enzyme is considered.

Amidohydrolases↗
PubMed1984

Silhouette photography of polyacrylamide slab gels.

A silhouette photography technique for making same-size negative films and positive contact prints of Coomassie blue-stained polyacrylamide slab gels is described. This technique does not require expensive photographic equipment and provides the resolution obtained with conventional camera systems.

Electrophoresis, Polyacrylamide Gel↗
PubMed1982

Probing the energetics of proteins through structural perturbation: sites of regulatory energy in human hemoglobin.

The sites of energy transduction within the human hemoglobin molecule for the regulation of oxygen affinity have been determined by an extensive study of the molecule's energetic response to structural alteration at individual amino acid residues. For 22 mutant and chemically modified hemoglobins we have determined the total free energy used by the tetrameric molecule for alteration of oxygen affinity at the four binding steps. The results imply that the regulation of oxygen binding affinity is due to energy changes which are mostly localized at the alpha 1 beta 2 interface. They also indicate a high degree of "internal cooperativity" within this contact region--i.e., the structural perturbations at individual residue sites are energetically coupled. Cooperativity in ligand binding is thus a reflection of cooperativity at a deeper level--that of the protein-protein interactions within the alpha 1 beta 2 interfacial domain.

Allosteric Regulation↗
PubMed1979

Binding of regulatory ligands to rabbit muscle phosphofructokinase. A model for nucleotide binding as a function of temperature and pH.

The binding of nucleoside triphosphates to rabbit muscle phosphofructokinase has been determined in 0.05 M phosphate buffers by changes in intrinsic protein fluorescence and by direct binding measurements. These experiments have been performed over a wide range of pH, temperature, and effector concentration. Quenching of protein fluorescence is shown to measure binding of nucleotides to a site which is not the active site but rather a site responsible for inhibition of the kinetic activity. This site is relatively specific for either ATP or MgATP with free ATP binding about 10-fold more tightly than MgATP. A model to describe binding to this site as a function of pH and temperature is proposed. This model assumes that the apparent affinity for ATP is determined by protonation of two ionizable groups (per subunit) and that ATP binds exclusively to protonated enzyme forms. Several ligands which affect the apparent affinity for nucleotide binding at the inhibitory site act by shifting the apparent pK of the ionizable groups. NH4+ and citrate do not influence nucleotide binding to the inhibitory site. At pH 6.9 in 0.05 M phosphate, low concentrations of MgATP or MgGTP enhance the protein fluorescence due to binding at the active site. The fluorescence studies and direct binding studies show that there is one active site and one inhibitory site per subunit. As described elsewhere (Pettigrew, D. W., and Frieden, C. (1978) J. Biol. Chem. 253, 3623-3627), there is a third nucleotide binding site on each subunit which is specific for cAMP, AMP, and ADP.

Adenine Nucleotides↗
PubMed1978

Rabbit muscle phosphofructokinase. Modification of molecular and regulatory kinetic properties with the affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine.

The affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine modifies rabbit muscle phosphofructokinase to the extent of one group/subunit. Modification appears to occur at a binding site specific for AMP, cyclic AMP, and ADP, i.e. those adenine nucleotides which are activators under conditions where regulatory kinetic behavior is obtained. The consequences of the modification are consistent with the model proposed previously for correlation between the pK of specific ionizable groups, regulatory kinetic behavior, ligand binding, and the reversible cold inactivation of the enzyme (Frieden, C., Gilbert. H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647). Thus, the modification shifts the apparent pK of the essential ionizable groups from 6.9 to 6.4 at 25 degrees C, with the result that regulatory kinetic behavior at pH 6.9 and 25 degrees C is lost. Furthermore, the apparent affinity of a site (other than the active site) for ATP, as measured by ATP-dependent quenching of intrinsic protein fluorescence at pH 6.9 and 25 degrees C, is decreased by the modification. Regulatory kinetic behavior for both substrates is obtained with the modified enzyme at a lower pH, consistent with the downward shift in the pK of the ionizable groups, but sensitivity to cAMP activation is abolished by the modification. The loss of regulatory kinetic behavior upon modification of sulfhydryl groups does not appear to be the same as that due to modification by the affinity label.

Adenosine↗
PubMed1978

Cleavage of native type III collagen in the collagenase susceptible region by thermolysin.

Viscometric assays were used to demonstrate the activity of thermolysin (EC 3.4.24.4) on native type III collagen in solution. Analysis of the reaction products by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electron microscopic visualisation of segment long spacing aggregates demonstrated localised cleavage of the collagen in the collagenase susceptible region.

Collagen↗