Biomedical subjects

D W Pettigrew

Publications and source records attributed to D W Pettigrew.

At least 19 recordsLinked to original sources

PubMed2001

IIA(Glc) allosteric control of Escherichia coli glycerol kinase: binding site cooperative transitions and cation-promoted association by Zinc(II).

The catalytic activity of glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inhibited allosterically by IIA(Glc) (previously known as III(Glc)), the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system. A sequentially contiguous portion of glycerol kinase undergoes an induced fit conformational change involving coil, alpha-helix, and 3(10)-helix upon IIA(Glc) binding. A second induced fit occurs upon binding of Zn(II) to a novel intermolecular site, which increases complex stability by cation-promoted association. Eight of the ten sequentially contiguous amino acids are substituted with alanine to evaluate the roles of these positions in complex formation. Effects of the substitutions reveal both favorable and antagonistic contributions of the normal amino acids to complex formation, and Zn(II) reverses these contributions for two of the amino acids. The consequences of some of the substitutions for IIA(Glc) inhibition are consistent with changes in the intermolecular interactions seen in the crystal structures. However, for the amino acids that are located in the region that is alpha-helical in the absence of IIA(Glc), the effects of the substitutions are not consistent with changes in intermolecular interactions but with increased stability of the alpha-helical region due to the higher alpha-helix propensity of alanine. The reduced affinity for IIA(Glc) binding seen for these variants is consistent with predictions of Freire and co-workers [Luque, I., and Freire, E. (2000) Proteins: Struct., Funct., Genet. 4, 63-71]. These variants show also increased cation-promoted association by Zn(II) so that the energetic contribution of Zn(II) to complex formation is doubled. The similarity of effects of the alanine substitutions of the amino acids in the alpha-helical region for IIA(Glc) binding affinity and cation-promoted association by Zn(II) indicates that they function as a cooperative unit.

Allosteric Regulation↗

PubMed2001

Subcloning, expression, purification, and characterization of Haemophilus influenzae glycerol kinase.

Glycerol kinase (EC 2.7.1.30) is a bacterial sugar kinase and a member of the sugar kinase/actin/hsc-70 superfamily of enzymes. The enzyme from Escherichia coli is an allosteric regulatory enzyme whose activity is inhibited by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (previously termed III(Glc)). Comparison of its primary structure with that of the highly similar Haemophilus influenzae glycerol kinase reveals that the amino acid sequence for the binding site for FBP is conserved while the amino acid sequence for the binding site for IIA(Glc) contains differences that are predicted to prevent its inhibition. To test this hypothesis, the H. influenzae glpK gene was assembled from DNA library fragments and subcloned into pUC18. The enzyme is expressed at high levels in E. coli. It was purified to greater than 90% homogeneity by taking advantage of its solubility behavior in a procedure that requires no column chromatography. The initial-velocity kinetic parameters of the purified enzyme are similar to those of the E. coli glycerol kinase. The H. influenzae glycerol kinase is inhibited by FBP but not by IIA(Glc), in agreement with the prediction based on sequence comparison. Sedimentation velocity experiments reveal that inhibition of HiGK by FBP is associated with oligomerization, behavior which is similar to EcGK. The possibility of utilizing mutagenesis studies to exploit the high degree of similarity of these two enzymes to elucidate the mechanism of allosteric regulation by IIA(Glc) is discussed.

Amino Acid Sequence↗

PubMed2001

The cyclization of farnesyl diphosphate and nerolidyl diphosphate by a purified recombinant delta-cadinene synthase.

The first step in the conversion of the isoprenoid intermediate, farnesyl diphosphate (FDP), to sesquiterpene phytoalexins in cotton (Gossypium barbadense) plants is catalyzed by delta-cadinene (CDN) synthase. CDN is the precursor of desoxyhemigossypol and hemigossypol defense sesquiterpenes. In this paper we have studied the mechanism for the cyclization of FDP and the putative intermediate, nerolidyl diphosphate, to CDN. A purified recombinant CDN synthase (CDN1-C1) expressed in Escherichia coli from CDN1-C1 cDNA isolated from Gossypium arboreum cyclizes (1RS)-[1-2H](E, E)-FDP to >98% [5-2H]and [11-2H]CDN. Enzyme reaction mixtures cyclize (3RS)-[4,4,13,13,13-2H5]-nerolidyl diphosphate to 62.1% [8,8,15,15,15-2H5]-CDN, 15.8% [6,6,15,15,15-2H5]-alpha-bisabolol, 8.1% [6,6,15,15,15-2H5]-(beta)-bisabolene, 9.8% [4,4,13,13-2H4]-(E)-beta-farnesene, and 4.2% unknowns. Competitive studies show that (3R)-nerolidyl diphosphate is the active enantiomer of (3RS)-nerolidyl diphosphate that cyclized to CDN. The kcat/Km values demonstrate that the synthase uses (E,E)-FDP as effectively as (3R)-nerolidyl diphosphate in the formation of CDN. Cyclization studies with (3R)-nerolidyl diphosphate show that the formation of CDN, (E)-beta-farnesene, and beta-bisabolene are enzyme dependent, but the formation of alpha-bisabolol in the reaction mixtures was a Mg2+-dependent solvolysis of nerolidyl diphosphate. Enzyme mechanisms are proposed for the formation of CDN from (E,E)-FDP and for the formation of CDN, (E)-beta-farnesene, and beta-bisabolene from (3RS)-nerolidyl diphosphate. The primary structures of cotton CDN synthase and tobacco epi-aristolochene synthase show 48% identity, suggesting similar three-dimensional structures. We used the SWISS-MODEL to test this. The two enzymes have the same overall structure consisting of two alpha-helical domains and epi-aristolochene synthase is a good model for the structure of CDN synthase. Several amino acids in the primary structures of both synthases superimpose. The amino acids having catalytic roles in epi-aristochene synthase are substituted in the CDN synthase and may be related to differences in catalytic properties.

Binding Sites↗

PubMed2001

Reverse genetics of Escherichia coli glycerol kinase allosteric regulation and glucose control of glycerol utilization in vivo.

Reverse genetics is used to evaluate the roles in vivo of allosteric regulation of Escherichia coli glycerol kinase by the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (formerly known as III(glc)), and by fructose 1,6-bisphosphate. Roles have been postulated for these allosteric effectors in glucose control of both glycerol utilization and expression of the glpK gene. Genetics methods based on homologous recombination are used to place glpK alleles with known specific mutations into the chromosomal context of the glpK gene in three different genetic backgrounds. The alleles encode glycerol kinases with normal catalytic properties and specific alterations of allosteric regulatory properties, as determined by in vitro characterization of the purified enzymes. The E. coli strains with these alleles display the glycerol kinase regulatory phenotypes that are expected on the basis of the in vitro characterizations. Strains with different glpR alleles are used to assess the relationships between allosteric regulation of glycerol kinase and specific repression in glucose control of the expression of the glpK gene. Results of these studies show that glucose control of glycerol utilization and glycerol kinase expression is not affected by the loss of IIA(Glc) inhibition of glycerol kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is the dominant allosteric control mechanism, and glucose is unable to control glycerol utilization in its absence. Specific repression is not required for glucose control of glycerol utilization, and the relative roles of various mechanisms for glucose control (catabolite repression, specific repression, and inducer exclusion) are different for glycerol utilization than for lactose utilization.

Allosteric Regulation↗

PubMed2001

Unexpected presence of defective glpR alleles in various strains of Escherichia coli.

Alleles of glpR associated with the same GlpR(-) phenotype produce substitutions in different conserved portions of the glycerol 3-phosphate repressor which are not part of the helix-turn-helix motif. Analysis of the effects on growth and enzyme expression show that glucose repression of glycerol utilization is not dependent on a functional repressor.

Alleles↗

PubMed1999

Crystal structures of Escherichia coli glycerol kinase variant S58-->W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion.

Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site [Ormo, M., Bystrom, C., and Remington, S. J. (1998) Biochemistry 37, 16565-16572]. The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition. Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures. The conformational difference is produced by a approximately 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily. Two of the ATP analogues bound in nonproductive conformations in both subunits. However, beta, gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol. This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion.

Actins↗

PubMed1998

Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation.

BACKGROUND: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70. RESULTS: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. CONCLUSIONS: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation.

Alanine↗

PubMed1998

Cation-promoted association of Escherichia coli phosphocarrier protein IIAGlc with regulatory target protein glycerol kinase: substitutions of a Zinc(II) ligand and implications for inducer exclusion.

In Escherichia coli, inducer exclusion is one mechanism by which glucose prevents unnecessary expression of genes needed for metabolism of other sugars. The basis for this mechanism is binding of the unphosphorylated form of the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system, IIAGlc (also known as IIIGlc), to a variety of target proteins to prevent uptake or synthesis of the inducer. One of these target proteins is glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), which is inhibited by IIAGlc. Glycerol kinase is the only IIAGlc target protein for which the structure of the complex is known. Association of these two proteins forms an intermolecular binding site for Zn(II) with metal ligands contributed by each protein, and Zn(II) enhances IIAGlc inhibition [Feese, M., Pettigrew, D. W., Meadow, N. D., Roseman, S., and Remington, S. J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3544-3548]. Here, we show that the Zn(II) enhancement can be described quantitatively by a model with binding of Zn(II) to the complex with an apparent dissociation constant of less than 1 microM at pH 7.0 and 25 degreesC. Initial velocity studies show that IIAGlc is an uncompetitive inhibitor with respect to both substrates, and the mechanism of inhibition is not altered by Zn(II). The Zn(II)-liganding residue contributed by glycerol kinase (Glu478) is substituted by using site-directed mutagenesis to construct the enzymes E478C, E478D, E478H, and E478Q. The substitutions have only small effects on the inhibition by IIAGlc in the absence of Zn(II), the catalytic properties, or other allosteric regulation. However, all of the substitutions abolish the Zn(II) enhancement of IIAGlc inhibition, and the X-ray crystallographic structures of the complexes of IIAGlc with the E478C and E478H mutants show these substitutions abolish binding of Zn(II) to the intermolecular site. These results support the hypothesis that Zn(II) enhances the affinity for complex formation by binding at the intermolecular site, i.e., cation promoted association. The high affinity for Zn(II) binding to the complex and the ability of the other four amino acid residues to efficiently substitute for Glu478 in all functions except binding of Zn(II) suggest that cation promoted association of these two proteins may have a role in inducer exclusion in vivo.

Cations↗

PubMed1998

Conserved active site aspartates and domain-domain interactions in regulatory properties of the sugar kinase superfamily.

The structures of the sugar kinase/heat shock 70/actin superfamily of enzymes show that the active site is located in a deep cleft between two domains whose relative movement defines a domain closure conformational change thought to be involved in the catalytic and regulatory properties of members of the superfamily. To investigate the role of the domain closure in the regulatory behavior, site-directed mutagenesis is used to alter specific domain-domain interactions in Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase), a member of this superfamily. Two active site aspartate residues are conserved throughout the superfamily, one (Asp245 in glycerol kinase) which is proposed to act as a general base during catalysis and one (Asp10 in glycerol kinase) which interacts with the Mg(II) ion of the bound Mg(II)-nucleotide complex. Each of these residues participates in domain-domain interactions that are mediated by the bound substrates. The enzymes containing the substitutions Asp245 to Asn (D245N) or Asp10 to Asn (D10N) were purified by affinity chromatography, and the effects of the substitutions on the catalytic properties and regulation by the allosteric effectors, fructose 1,6-bisphosphate (FBP), and the glucose-specific phosphocarrier protein, IIIGlc (also known as IIAGlc), were determined. Each of the residues participates in catalysis; kcat/Katp is decreased 300-fold by the D245N substitution and 100-fold by the D10N substitution. Affinity labeling with the glycerol analog 1,3-dichloroacetone shows that the level of activity seen for the D245N mutant enzyme is not due to deamidation of the substituted asparagine. Each of the substitutions has little effect on regulation by FBP and the apparent affinity for IIIGlc, and the D245N substitution does not affect the extent of inhibition by IIIGlc. However, the D10N substitution decreases the maximum extent of inhibition by IIIGlc from 100 to 60%, thus changing the action of IIIGlc to that of a partial inhibitor. The different sensitivities of the extents of FBP and IIIGlc inhibition to perturbation of a domain-domain interaction mediated by Asp10 suggest that the relations of the actions of these allosteric effectors to the domain closure conformational change are different.

Allosteric Regulation↗

PubMed1996

A single amino acid change in Escherichia coli glycerol kinase abolishes glucose control of glycerol utilization in vivo.

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in glucose control of glycerol metabolism. Its catalytic activity is inhibited allosterically by the glycolytic intermediate, fructose 1,6-biphosphate, and by the phosphotransferase system phosphocarrier protein, IIIGlc (also known as IIAGlc). These inhibitors provide mechanisms by which glucose blocks glycerol utilization in vivo. We report here the cloning and sequencing of the glpK22 gene isolated from E. C. C. Lin strain 43, a strain that shows the loss of glucose control of glycerol utilization. DNA sequencing shows a single missense mutation that translates to the amino acid change Gly-304 to Ser (G-304-S) in glycerol kinase. The effects of this substitution on the functional and physical properties of the purified mutant enzyme were determined. Neither of the allosteric ligands inhibits it under conditions that produce strong inhibition of the wild-type enzyme, which is sufficient to explain the phenotype of strain 43. However, IIIGlc activates the mutant enzyme, which could not be predicted from the phenotype. In the wild-type enzyme, G-304 is located 1.3 nm from the active site and 2.5 nm from the IIIGlc binding site (M. Feese, D. W. Pettigrew, N. D. Meadow, S. Roseman, and S. J. Remington, Proc. Natl. Acad. Sci. USA 91:3544-3548, 1994). It is located in the same region as amino acid substitutions in the related protein DnaK which alter its catalytic and regulatory properties and which are postulated to interfere with a domain closure motion (A. S. Kamath-Loeb, C. Z. Lu, W.-C. Suh, M. A. Lonetto, and C. A. Gross, J. Biol. Chem. 270:30051-30059, 1995). The global effect of the G-304-S substitution on the conformation and catalytic and regulatory properties of glycerol kinase is consistent with a role for the domain closure motion in the molecular mechanism for glucose control of glycerol utilization.

Allosteric Regulation↗

PubMed1994

Escherichia coli glycerol kinase: role of a tetramer interface in regulation by fructose 1,6-bisphosphate and phosphotransferase system regulatory protein IIIglc.

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in a signal transduction pathway that couples expression of genes required for glycerol metabolism to the relative availability of glycerol and glucose. Its catalytic activity is inhibited by protein-protein interactions with IIIglc, a phosphotransferase system protein, and by fructose 1,6-bisphosphate (FBP); each of these allosteric effectors constitutes a positive signal that glucose is available. Loss of glucose inhibition of glycerol metabolism was used to screen for regulatory mutants of glycerol kinase after hydroxylamine mutagenesis of the cloned glpK gene. Two mutant enzymes were identified and shown by DNA sequencing to contain the mutations alanine 65 to threonine (A65T) and aspartate 72 to asparagine (D72N). Initial velocity studies show the mutations do not significantly affect the catalytic properties, hence active-site structures, of the enzymes. Both mutations decrease inhibition by FBP; A65T eliminates the inhibition while D72N appears to decrease the affinity for FBP and the extent of the inhibition. However, neither mutation significantly affects inhibition by IIIglc. Gel-permeation chromatography studies show that both of the mutations alter the dimer-tetramer assembly reaction of the enzyme and the effect of FBP in increasing the molecular weight. The effects of the mutations on the assembly reaction are consistent with the locations of these two amino acid residues in the X-ray structure, which shows them to be associated with an alpha-helix that constitutes one of the two subunit-subunit interfaces within the tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1994

Cation-promoted association of a regulatory and target protein is controlled by protein phosphorylation.

A central question in molecular biology concerns the means by which a regulatory protein recognizes different targets. IIIGlc, the glucose-specific phosphocarrier protein of the bacterial phosphotransferase system, is also the central regulatory element of the PTS. Binding of unphosphorylated IIIGlc inhibits several non-PTS proteins, but there is little or no sequence similarity between IIIGlc binding sites on different target proteins. The crystal structure of Escherichia coli IIIGlc bound to one of its regulatory targets, glycerol kinase, has been refined at 2.6-A resolution in the presence of products, adenosine diphosphate and glycerol 3-phosphate. Structural and kinetic analyses show that the complex of IIIGlc with glycerol kinase creates an intermolecular Zn(II) binding site with ligation identical to that of the zinc peptidase thermolysin. The zinc is coordinated by the two active-site histidines of IIIGlc, a glutamate of glycerol kinase, and a water molecule. Zn(II) at 0.01 and 0.1 mM decreases the Ki of IIIGlc for glycerol kinase by factors of about 15 and 60, respectively. The phosphorylation of one of the histidines of IIIGlc, in its alternative role as phosphocarrier, provides an elegant means of controlling the cation-enhanced protein-protein regulatory interaction. The need for the target protein to supply only one metal ligand may account for the lack of sequence similarity among the regulatory targets of IIIGlc.

Binding Sites↗

PubMed1993

Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol kinase.

The phosphocarrier protein IIIGlc is an integral component of the bacterial phosphotransferase (PTS) system. Unphosphorylated IIIGlc inhibits non-PTS carbohydrate transport systems by binding to diverse target proteins. The crystal structure at 2.6 A resolution of one of the targets, glycerol kinase (GK), in complex with unphosphorylated IIIGlc, glycerol, and adenosine diphosphate was determined. GK contains a region that is topologically identical to the adenosine triphosphate binding domains of hexokinase, the 70-kD heat shock cognate, and actin. IIIGlc binds far from the catalytic site of GK, indicating that long-range conformational changes mediate the inhibition of GK by IIIGlc. GK and IIIGlc are bound by hydrophobic and electrostatic interactions, with only one hydrogen bond involving an uncharged group. The phosphorylation site of IIIGlc, His90, is buried in a hydrophobic environment formed by the active site region of IIIGlc and a 3(10) helix of GK, suggesting that phosphorylation prevents IIIGlc binding to GK by directly disrupting protein-protein interactions.

Adenosine Diphosphate↗

PubMed1992

Mutagenic dissection of hemoglobin cooperativity: effects of amino acid alteration on subunit assembly of oxy and deoxy tetramers.

Free energies of oxygen-linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (2) Structural localization of cooperative free energy. Difference free energies of dimer-tetramer assembly (oxy minus deoxy) yielded delta Gc for each hemoglobin, i.e., the free energy used for modulation of oxygen affinity over all four binding steps. A structure-energy map constructed from these results shows that the alpha 1 beta 2 interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated dimers of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to dimer dissociation. (4) Additivity, site-site coupling and adventitious perturbations. All these are exhibited by individual-site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change. (5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side-chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms.

Hemoglobins↗

PubMed1990

Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies.

Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) was investigated by using both kinetics and binding methods. Initial-velocity studies in both reaction directions show a sequential kinetic mechanism with apparent substrate activation by ATP and substrate inhibition by ADP. In addition, the Michaelis constants differ greatly from the substrate dissociation constants. Results of product inhibition studies and dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme has a random kinetic mechanism, which is consistent with the observed formation of binary complexes with all the substrates and the glycerol-independent MgATPase activity of the enzyme. Dissociation constants for substrate binding determined by using ligand protection from inactivation by N-ethylmaleimide agree with those estimated from the initial-velocity studies. Determinations of substrate binding stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP, ADP, and glycerol. Thus, the regulation by nucleotides does not appear to reflect binding at a separate regulatory site. The random kinetic mechanism obviates the need to postulate such a site to explain the formation of binary complexes with the nucleotides. The observed stoichiometry is consistent with a model for the nucleotide regulatory behavior in which the dimer is the enzyme form present in the assay and its subunits display different substrate binding affinities. Several properties of the enzyme are consistent with negative cooperativity as the basis for the difference in affinities. The possible physiological importance of the regulatory behavior with respect to ATP is considered.

Adenosine Diphosphate↗

PubMed1989

Crystallization and preliminary X-ray studies of Escherichia coli glycerol kinase.

Escherichia coli glycerol kinase, a major regulatory enzyme which catalyzes the reversible MgATP-dependent phosphorylation of glycerol has been crystallized by the hanging drop vapor diffusion method at room temperature. Three different crystal forms have been obtained in the presence of glycerol and appear to be suitable for X-ray crystallographic studies. Vapor diffusion against 55% ammonium sulfate and 1% beta-octyl glucoside (pH 7.0) yields rhombohedral crystals with space group R32, a = b = 277.1 A, c = 78.7 A (hexagonal indexing) containing a dimer of Mr 112,000 in the asymmetric unit (Vm = 2.64 A3/dalton). Vapor diffusion against sodium chloride in the presence of 10% (w/v) polyethylene glycol (pH 6.5 to 7.0) yields two different crystal forms, both with space group P2(1). The first form has a = 88.1 A, b = 99.3 A, c = 114.6 A, beta = 119 degrees, the second form has a = 92.5 A, b = 117.6 A, c = 108.3 A, beta = 93.64 degrees. Addition of ADP enhances growth of the monoclinic forms. These forms appear to contain an entire tetramer of Mr 224,000 in the asymmetric unit and have Vm values of 2.28 and 2.65 A3/dalton, respectively. All forms diffract to better than 3.0 A resolution while the second monoclinic form diffracts to approximately 1.8 A.

Crystallization↗

PubMed1989

Near-fatal hydrogen peroxide ingestion.

Fatal or near-fatal hydrogen peroxide ingestions are rarely encountered. We report the case of a 33-year-old woman who experienced seizure activity, respiratory arrest, and residual neurologic deficits after accidentally ingesting industrial-strength 35% hydrogen peroxide.

Administration, Oral↗

PubMed1988

Escherichia coli glycerol kinase. Cloning and sequencing of the glpK gene and the primary structure of the enzyme.

The glpK gene, which codes for Escherichia coli K-12 glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), has been cloned into the HindIII site of pBR322. The gene was contained in a 2.8-kilobase DNA fragment which was obtained from a lambda transducing bacteriophage, lambda dglpK100 (Conrad, C.A., Stearns, G.W., III, Prater, W.E., Rheiner, J.A., and Johnson, J.R. (1984) Mol. Gen. Genet. 195, 376-378). The DNA sequence of 2 kilobases of the cloned HindIII fragment was obtained using the dideoxynucleotide method. The start of the open reading frame for the glpK gene was identified from the N-terminal sequence of the first 22 amino acid residues of the purified enzyme, which was determined by automated Edman degradation. The open reading frame codes for a protein of 502 amino acids and a molecular weight of 56,106 which is in good agreement with the value previously determined by sedimentation equilibrium. The primary structure of the protein as deduced from the gene sequence was corroborated by the isolation and sequencing of four tryptic peptides, which were found to occur at the following amino acid locations: 173-177, 203-211, 279-281, 464-468. The N-terminal sequence of the purified enzyme shows that the enzyme undergoes post-translational processing. Restriction digestion as well as DNA sequencing of the supercoiled plasmid shows that the HindIII fragment is inserted into pBR322 such that the glpK gene is transcribed in a counterclockwise direction. Examination of the upstream DNA sequence reveals two possible promoters of essentially the same efficiency: the P1 promoter of pBR322 and a hybrid promoter which contains both bacterial and pBR322 DNA sequences.

Alleles↗