Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

African trypanosomiasis, sleeping sickness in humans or nagana in animals, is a potentially fatal neglected tropical disease and a threat to 65 million human lives and 100 million small and large livestock animals in sub-Saharan Africa. Available treatments for this devastating disease are few and have limited efficacy, prompting the search for new drug candidates. Simultaneous inhibition of the trypanosomal glycerol kinase (TGK) and trypanosomal alternative oxidase (TAO) is considered a validated strategy toward the development of new drugs. Our goal is to develop a TGK-specific inhibitor for coadministration with ascofuranone (AF), the most potent TAO inhibitor. Here, we report on the identification of novel compounds with inhibitory potency against TGK. Importantly, one of these compounds (compound 17) and its derivatives (17a and 17b) killed trypanosomes even in the absence of AF. Inhibition kinetics revealed that derivative 17b is a mixed-type and competitive inhibitor for TGK and TAO, respectively. Structural data revealed the molecular basis of this dual inhibitory action, which, in our opinion, will aid in the successful development of a promising drug to treat trypanosomiasis. Although the EC50 of compound 17b against trypanosome cells was 1.77 µM, it had no effect on cultured human cells, even at 50 µM.-Balogun, E. O., Inaoka, D. K., Shiba, T., Tsuge, C., May, B., Sato, T., Kido, Y., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Michels, P. A. M., Watanabe, Y.-I., Moore, A. L., Harada, S., Kita, K. Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

Aberrant expression of miR-451a contributes to 1,2-dichloroethane-induced hepatic glycerol gluconeogenesis disorder by inhibiting glycerol kinase expression in NIH Swiss mice.

The identification of aberrant microRNA (miRNA) expression during chemical-induced hepatic dysfunction will lead to a better understanding of the substantial role of miRNAs in liver diseases. 1,2-Dichloroethane (1,2-DCE), a chlorinated organic toxicant, can lead to hepatic abnormalities in occupationally exposed populations. To explore whether aberrant miRNA expression is involved in liver abnormalities mediated by 1,2-DCE exposure, we examined alterations in miRNA expression patterns in the livers of NIH Swiss mice after dynamic inhalation exposure to 350 or 700 mg m-3 1,2-DCE for 28 days. Using a microarray chip, we discovered that only mmumiR-451a was significantly upregulated in the liver tissue of mice exposed to 700 mg m-3 1,2-DCE; this finding was validated by quantitative real-time polymerase chain reaction. In vitro study revealed that it was metabolite 2-chloroacetic acid, not 1,2-DCE that resulted in the upregulation of mmu-miR-451a in the mouse AML12 cell line. Furthermore, our data showed that the upregulation of mmu-miR-451a induced by 2-chloroacetic acid could suppress the expression of glycerol kinase and lead to the inhibition of glycerol gluconeogenesis in mouse liver tissue and AML12 cells. These observations provide evidence that hepatic mmu-miR-451a responds to 1,2-DCE exposure and might induce glucose metabolism disorders by suppressing the glycerol gluconeogenesis process.

MicroRNA-645 promotes cell metastasis and proliferation of renal clear cell carcinoma by targeting GK5.

OBJECTIVE: To dissect the functioning mode of miR-645 on renal clear cell carcinoma cell metastasis and growth, and provide therapeutic targets for renal clear cell carcinoma. PATIENTS AND METHODS: Quantitative Real-time PCR (qRT-PCR) assay was employed to detect miR-645 expression level. Wound healing assay and transwell assay were performed to investigate metastasis capacity of renal clear cell carcinoma cells. Cell Counting Kit 8 (CCK8) assay was incorporated to assess cell proliferation capacity. Flow cytometry was used to identify cell apoptosis and cell cycle distribution. Protein levels were assessed by Western blotting assay. The target gene was predicted and verified by bioinformatics analysis and luciferase assay. RESULTS: MiR-645 was upregulated in renal clear cell carcinoma tissues when compared with para-carcinoma tissues (n=32). Downregulated miR-645 could attenuate cell migration and invasion capacities, as well as inhibited cell proliferation capacity, promoted cell apoptosis and cell cycle arrest at G0/G1 phase. GK5 was chosen as the target gene of miR-645 by bioinformatics analysis and luciferase reporter assay. Moreover, silence of GK5 could rescue tumor suppression role of downregulated miR-645 on renal clear cell carcinoma metastasis. CONCLUSIONS: Knockdown of miR-645 exerted tumor-suppressive effects on renal clear cell carcinoma metastasis and growth via targeting GK5 in vitro, which provided an innovative and candidate target for diagnose and treatment of renal clear cell carcinoma.

Biochemical characterization of highly active Trypanosoma brucei gambiense glycerol kinase, a promising drug target.

Human African trypanosomes are blood parasites that cause sleeping sickness, a debilitating disease in sub-Saharan Africa. Glycerol kinase (GK) of these parasites additionally possesses a novel property of reverse catalysis. GK is essential to blood stream form trypanosome, and therefore a promising drug target. Here, utilizing recombinant DNA technology an optimized procedure for obtaining large amount of the purified protein was established. Furthermore, biochemical data on its enzymology are reported. The protein was maximally active at pH 6.8 over a temperature range of 25-70°C, with activation energy of 34.02 ± 0.31 kJ mol(-1). The enzyme catalyses a reversible bisubstrate [ADP and glycerol 3-phosphate (G3P)]-biproduct (ATP and glycerol) reaction. It has Km of 0.90 and 5.54 mM for ADP and G3P, respectively, and Vmax of 25.3 and 20.0 µmol min(-1) mg(-1), respectively. Unexpectedly, the enzyme lost more than 50% of its activity in 48 h at 4°C in 0.1 M sodium phosphate buffer pH 6.8 containing 10 mM MgSO4. However, perfect stabilization of the GK for more than 4 weeks was achieved in the presence of its natural ligands and cofactor. Using this stabilized protein, crystals of trypanosome GK with better resolution were obtained. This will accelerate the success of GK inhibitor development for drug design.

Naphthoquinone derivatives exert their antitrypanosomal activity via a multi-target mechanism.

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Oligomeric interactions provide alternatives to direct steric modes of control of sugar kinase/actin/hsp70 superfamily functions by heterotropic allosteric effectors: inhibition of E. coli glycerol kinase.

Unlike those for monomeric superfamily members, heterotropic allosteric effectors of the tetrameric Escherichia coli glycerol kinase (EGK) bind to only one of the two domains that define the catalytic cleft and far from the active site. An R369A amino acid substitution removes oligomeric interactions of a novel mini domain-swap loop of one subunit with the catalytic site of another subunit, and an A65T substitution perturbs oligomeric interactions in a second interface. Linked-functions enzyme kinetics, analytical ultracentrifugation, and FRET are used to assess effects of these substitutions on the allosteric control of catalysis. Inhibition by phosphotransferase system protein IIA(Glc) is reduced by the R369A substitution, and inhibition by fructose 1,6-bisphosphate is abolished by the A65T substitution. The oligomeric interactions enable the heterotropic allosteric effectors to act on both domains and modulate the catalytic cleft closure despite binding to only one domain.

IIAGlc inhibition of glycerol kinase: a communications network tunes protein motions at the allosteric site.

Steady-state and time-resolved fluorescence anisotropy methods applied to an extrinsic fluorophore that is conjugated to non-native cysteine residues demonstrate that amino acids in an allosteric communication network within a protein subunit tune protein backbone motions at a distal site to enable allosteric binding and inhibition. The unphosphorylated form of the phosphocarrier protein IIAGlc is an allosteric inhibitor of Escherichia coli glycerol kinase, binding more than 25 A from the kinase active site. Crystal structures that showed a ligand-dependent conformational change and large temperature factors for the IIAGlc-binding site on E. coli glycerol kinase suggest that motions of the allosteric site have an important role in the inhibition. Three E. coli glycerol kinase amino acids that are located at least 15 A from the active site and the allosteric site were shown previously to be necessary for transplanting IIAGlc inhibition into the nonallosteric glycerol kinase from Haemophilus influenzae. These three amino acids are termed the coupling locus. The apparent allosteric site motions and the requirement for the distant coupling locus to transplant allosteric inhibition suggest that the coupling locus modulates the motions of the IIAGlc-binding site. To evaluate this possibility, variants of E. coli glycerol kinase and the chimeric, allosteric H. influenzae glycerol kinase were constructed with a non-native cysteine residue replacing one of the native residues in the IIAGlc-binding site. The extrinsic fluorophore Oregon Green 488 (2',7'-difluorofluorescein) was conjugated specifically to the non-native cysteine residue. Steady-state and time-resolved fluorescence anisotropy measurements show that the motions of the fluorophore reflect backbone motions of the IIAGlc-binding site and these motions are modulated by the amino acids at the coupling locus.

A novel pathway for lipid biosynthesis: the direct acylation of glycerol.

The acylation of glycerol-3-phosphate by acyl-CoA is regarded as the first committed step for the synthesis of the lipoidal moiety in glycerolipids. The direct acylation of glycerol in mammalian tissues has not been demonstrated. In this study, lipid biosynthesis in myoblasts and hepatocytes was reassessed by conducting pulse-chase experiments with [1,3-(3)H]glycerol. The results demonstrated that a portion of labeled glycerol was directly acylated to form monoacylglycerol and, subsequently, diacylglycerol and triacylglycerol. The direct acylation of glycerol became more prominent when the glycerol-3-phosphate pathway was attenuated or when exogenous glycerol levels became elevated. Glycerol:acyl-CoA acyltransferase activity, which is responsible for the direct acylation of glycerol, was detected in the microsomal fraction of heart, liver, kidney, skeletal muscle, and brain tissues. The enzyme from pig heart microsomes displayed optimal activity at pH 6.0 and the preference for arachidonyl-CoA as the acyl donor. The apparent K(m) values for glycerol and arachidonyl-CoA were 1.1 mM and 0.17 mM, respectively. The present study demonstrates the existence of a novel lipid biosynthetic pathway that may be important during hyperglycerolemia produced in diabetes or other pathological conditions.

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.

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.

Crystal structure of a complex of Escherichia coli glycerol kinase and an allosteric effector fructose 1,6-bisphosphate.

The three-dimensional structures of Escherichia coli glycerol kinase (GK) with bound glycerol in the presence and absence of one of the allosteric regulators of its activity, fructose 1,6-bisphosphate (FBP), at 3.2 and 3.0 A, are presented. The molecule crystallized in space group P41212, and the structure was solved by molecular replacement. The models were refined with good stereochemistry to final R-factors of 21.1 and 21.9%, respectively. A tetrameric arrangement of monomers was observed which was essentially identical to the proposed inactive tetramer II previously described [Feese, M. D., Faber, H. R., Bystrom, C. E., Pettigrew, D. W., and Remington, S. J. (1998) Structure (in press)]. However, the crystal packing in this form was especially open, permitting the FBP binding site to be occupied and identified. The crystallographic data revealed a most unusual type of FBP binding site formed between two glycine-arginine loops (residues 234-236) where one-half of the binding site is donated by each monomer at the regulatory interface. The molecule of FBP binds in two mutually exclusive modes on a noncrystallographic 2-fold axis at 50% occupancy each; thus, a tetramer of GK binds two molecules of FBP. Ionic interactions between the 1- and 6-phosphates of FBP and Arg 236 were observed in addition to hydrogen bonding interactions between the backbone amide of Gly 234 and the 6-phosphate. No contacts between the protein and the furanose ring were observed. Mutagenesis of Arg 236 to alanine drastically reduced the extent of inhibition of GK by FBP and lowered, but did not eliminate, the ability of FBP to promote tetramer association. These observations are consistent with the previously characterized mechanism of FBP inhibition of GK, in which FBP acts both to promote dimer-tetramer assembly and to inactivate the tetramers.

Characterization of glycerol uptake and glycerol kinase activity in rat hepatocytes cultured under different hormonal conditions.

Glycerol uptake and glycerol kinase activity were studied in primary cultures of rat hepatocytes in the presence of either 1 nM insulin, 1 nM glucagon, or 100 nM dexamethasone, alone or in combination in the culture medium. Glycerol uptake exhibited saturation kinetic with K(m) values (microM) and Vmax (nmol/min x mg protein) ranging from 250-402, and 7.9-10.1, respectively. The corresponding K(m) and Vmax values for glycerol kinase activity were 36-46 and 8.7-12.7. Using the metabolic uncoupler 2,4-dinitrophenol, glycerol uptake and the cellular content of glycerol phosphorylated metabolites were reduced 33% and 43%, respectively, whereas no decrease in the cellular content of glycerol was seen. The glycerol analogues monoacetin, monobutyrin and dihydroxypropyl dichloroacetate were able in a concentration-dependent manner to inhibit glycerol uptake into hepatocytes with the two latter having IC50 values of approximately 1 mM. Moreover, it was demonstrated that the three glycerol analogues were substrates for glycerol kinase, which indicates a competitive mode of inhibition. The kinetic parameters for these substrates were calculated by using glycerol kinase from Candida Mycoderma. Monobutyrin was found to be 4 times lees efficient as substrate compared to the other substrates. Overall, these results indicate that independently of the culture conditions, glycerol uptake is the rate-limiting step in glycerol metabolism, and that the investigated glycerol analogues are metabolized via the same route as glycerol.

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.

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.

Selective interaction of glycosomal enzymes from Trypanosoma brucei with hydrophobic cyclic hexapeptides.

Hydrophobic cyclic hexapeptides have been reported to selectively inhibit glycosomal triosephosphate isomerase from Trypanosoma brucei (Kuntz et al, 1992, Eur. J. Biochem., 207, 441-447). Here it is shown that this inhibition is not due to a specific interaction between the enzyme and soluble hydrophobic cyclic hexapeptides, but that it is the result of a coprecipitation of trypanosome triosephosphate isomerase with cyclic hexapeptides when the solubilities of the latter are exceeded. A study of the interaction of these hexapeptides with other glycosomal enzymes revealed that several of them, such as phosphoglycerate kinase and hexokinase, also coprecipitated with these peptides, whereas most of the homologous enzymes from other organisms did not coprecipitate, nor were they inactivated.

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.

The inhibition of glucokinase and glycerokinase from Bacillus stearothermophilus by the triazine dye Procion Blue MX-3G.

Glucokinase from Bacillus stearothermophilus was irreversibly inactivated by the reactive dichlorotriazinyl dye Procion Blue MX-3G at pH 8.0. The enzyme was protected from inactivation by the substrate MgATP. Kinetic data implied that the dye occupied the MgATP-binding site. The apparent Km values for MgATP and D-glucose were found to be 70 microM and 210 microM respectively, and the Kd of the pure reactive dye was 16 microM; 1 mol of the pure reactive dye bound to 1 mol of glucokinase subunit. The dye was shown to have potential as an affinity probe for glucokinase. Glycerokinase from the same bacterium was inactivated by Procion Blue MX-3G at high concentrations (5 mM), but only after a period of increased enzyme activity. Kinetic data indicated that the dye preferentially attacked the glycerol-binding site. The apparent Km values for MgATP and glycerol were found to be 38 microM and 13 microM respectively, and 4 mol of reactive dye could be bound to 1 mol of glycerokinase subunit. This was surprising in view of the MgATP-dependent elution of glycerokinase from immobilized Procion Blue MX-3G.

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.

1-Thioglycerol: inhibitor of glycerol kinase activity in vitro and in situ.

The infantile form of glycerol kinase (GK) deficiency (McKusick No. 30703) (1) is characterized by adrenal cortical insufficiency, adrenal hypoplasia and developmental delay. The underlying biochemical mechanism(s) responsible for the observed clinical presentations are undetermined. Pursuant to our examination of the molecular pathogenesis of this enzyme deficiency, we have endeavored to develop a model for this disorder. 1-thioglycerol (1-TG) was investigated as a potential GK inhibitor in adrenal gland, an organ consistently affected, and in cultured fibroblasts, available from affected individuals. In 105,000 g bovine adrenal supernatant the Ki for 1-TG was 1.9 mM. In human fibroblast 105,000 g supernatant, the Ki for 1-TG was 3.4 mM. In both tissues the inhibition was purely competitive with respect to glycerol. Using incorporation of [14C(U)]-glycerol into protein as an index of GK activity in situ in human skin fibroblasts, GK deficient fibroblasts incorporate less than 10% of that observed in normal fibroblasts. Addition of 1-TG to normal fibroblasts resulted in inhibited incorporation rates. The specificity of these effects in situ was examined. Our findings indicate that 1-TG may be a suitable inhibitor of GK activity for the development of a model for glycerol kinase deficiency.