Stoichiometry of labeling of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate.

A site-specific analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate (S2P-PNP), inactivates the ATPase activities of myosin's proteolytic fragments, heavy meromyosin (HMM) and subfragment one (SF1), by formation of mixed disulfides between the 6 position of the purine ring and certain key cysteines. The stoichiometry of the reaction was determined by quantitatively displacing the thiopurine nucleotides from the labeled enzymes with sodium[14-C]cyanide. The thiocyanatoenzyme formed regained 25 percent of the original activity showing that the cysteines modified were not essential for catalysis. The rate of uptake of label paralleled the rate of inactivation. HMM was completely inactivated when 4 mol of thiopurine nucleotide was bound. SF1 made by a papain digestion of myosin incorporarted 2 mol of thiopurine nucleotide when completely inactivated. Having adenylyl imidodiphosphate, areversible competitive inhibitor of myosin's ATPase, present during the inactivation of HMM by S2P-PNP demonstrated that only one cysteine per head needed to be blocked to inactivate the enzyme. Moreover, SF1 made by a trypsin digest of HMM was completely inactivated when only 1.1 mol of the thiopurine nucleotide bound again indicating that blocking only a single cysteine per head was sufficient to cause inactivation. This sulfhydryl is thought to be at an ATP binding site distinct from the ATPase site. The properties of this second ATP binding site are consistent with it being an ATP regulatory site.     

Reaction of cardiac myosin with a purine disulfide analog of adenosine triphosphate. I. Kinetics of inactivation and binding of adenylyl imidodiphosphate.

Bovine cardiac myosin ATPase activity was rapidly inactivated by the purine disulfide analog of ATP,6,6'-dithiobis(inosinyl imidodiphosphate). Kinetic investigations showed that this analog acted as a site-specific reagent at 0 degrees with a Ki of 130 muM and a half-life of 8.2 min at saturating inhibitor concentrations. Concentrations (50 to 500 muM) of ATP, adenyl-5'-yl imidodiphosphate (AMP-PNP), or ADP that saturated the active site caused an enhancement in the rate of inactivation, indicating the purine disulfide analog was not reacting at the active site. Under these conditions saturation kinetic data were still observed with Ki values remaining unchanged (120 muM) but with the half-life of inactivation decreasing to 6.0 min (ATP) and 4.6 min (AMP-PNP) at saturating inhibitor concentrations. At concentrations greater than 0.5 mM ATP, AMP-PNP, or ADP there was a decrease in the rate of inactivation, implying protection by these nucleotides. However, saturation kinetics of inactivation could no longer be demonstrated, implying a change in the mechanism of inactivation. A comparison of the inactivation of the Mg2+, Ca2+, and EDTA-ATPase activities of cardiac myosin after modification by the purine disulfide analog showed that the Mg2+- and Ca2+ATPase activities plateaued at approximately 60% and 40%, respectively, while the EDTA-ATPase activity continued to decrease to below 10%. This evidence supports the suggestion that the purine disulfide analog was not reacting at the active site. Equilibrium dialysis experiments were used to measure the binding of [8-3H]AMP-PNP to native cardiac myosin, the thiopurine nucleotide-modified myosin, and the derivative formed by displacing the thiopurine nucleotide by cyanide (thiocyanato-myosin). Native myosin bound a total of 2.1 mol of AMP-PNP with a binding constant of 6.0 X 10(6) M-1. There was a 15 to 40% decrease in the number of AMP-PNP binding sites in the enzyme derivatives, but the active sites appeared not to be blocked since the association constants remained essentially unchanged (KA=3.9 X 10(6) M-1 for thiopurine nucleotide-myosin and 12.0 X 10(6) M-1 for thiocyanato-myosin). The kinetic studies and the binding experiments indicate that the purine disulfide analog reacts at a specific site other than the active site but do not offer support to earlier suggestions from skeletal myosin studies that this site is a possible ATP control site.     

Reaction of cardiac myosin with a purine disulfide analog of adenosine triphosphate. II. Stoichiometry and subunit location of labeling.

The reaction of bovine cardiac myosin with the site-specific purine disulfide analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate), was studied to determine the stoichiometry of labeling and subunit location of the reactive cysteines. The analog inactivates myosin by forming a mixed disulfide between the thiopurine nucleotide and certain key cysteines. The thiopurine nucleotide was displaced quantitatively by 14CN to form the more stable thiocyanato-enzyme derivatives. In cardiac myosin, the reactive cysteines could be categorized into three classes, nonessential, critical, and noncritical. The modification of the critical cysteines (two per myosin) inactivated the EDTA and Ca2+ ATPase activities, the latter to a lesser extent. The nonessential cysteines (two to three per myosin) and the noncritical cysteines (two per myosin), differentiated by their rates of reaction, had no effect on the ATPase activities after modification. Thiocyanato-modified myosin was analyzed by sodium dodecyl sulfate gel electrophoresis to determine the distribution of 14CN in the subunits. The critical cysteines were found on the 21,000-dalton light chain (LC1) and the noncritical cysteines on the heavy chains. More specifically, the critical cysteine modified in cardiac LC1 (determined from the products after cyclization and chain cleavage at the thiocyanatoalanyl residues) was shown to be the thiol residue whose surrounding amino acid sequence is homologous to that of the single cysteine of the skeletal myosin alkali light chains, confirming the likely similar structure and function of these light chains in the two different muscle types.     

Subunit location of sulfhydryl groups of myosin labeled with a purine disulfide analog of adenosine triphosphate.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfides with cysteine residues at what are believed to be ATP regulatory sites of myosin. Blocking these sites causes inactivation of the ATPase activity at the active sites. Two cysteine residues per head are specifically modifed by this disulfide analog. The thiopurine nucleotides can be stoichiometrically displaced from myosin by [14-C]cyanide to give a more stable thiocyanato derivative of the enzyme. [14-C]Thiocyanatomyosin (3.7 14-CN/myosin) was dissociated in 4 M urea and the individual subunits were isolated. The heavy chains each had 0.78 14-CN bound per 200,000 molecular weight unit. The light chain with molecular weight of 20,700 had 1.00 14-CN bound and the 16,500 molecular weight light chain had 0.65 14-CN bound. The two 19,000 molecular weight light chains were not labeled. The two labeled light chains have only a single cysteine which is stoichiometrically modified. These two light chains show a high degree of homology and presumably perform identical functions in myosin. Their specific modification by the purine disulfide analog and their other known properties suggest that they contribute directly to the ATP regulatory sites and may, in fact, function as regulatory subunits.     

A novel adenosine triphosphate analog with a heavy atom to target the nucleotide binding site of proteins.

We have synthesized 2'-deoxy-2'-iodoadenosine-5'-triphosphate (2'-IATP), a heavy-atom analog of adenosine-5'-triphosphate. This compound was made for X-ray structural studies to target the nucleotide site of ATP binding proteins. It was diffused successfully into crystals of the microtubule-based motor proteins ncd (non-claret disjunctional protein from Drosophila melanogaster) and kinesin. With ncd, the nucleotide binding site was 70% occupied and the crystals were able to diffract X-rays to 2.5 A. The iodo-analog provided a useful isomorphous derivative with overall phasing power 1.89 in the range of 25.0-2.5 A. With kinesin, 2'-IATP co-crystallized with the protein. The crystals diffracted to at least 2.8 A with a phasing power of 1.73 in the range of 20.0-5.0 A. The analog was also found to be a substrate for all of the enzymes tested, including creatine kinase, pyruvate kinase, hexokinase, and myosin, with values of Km and Vmax that were within a factor of 10 of those for ATP. The analog supported muscle contraction, relaxing fibers, and producing active tension with values not statistically different from those obtained with ATP. These results all suggest that this analog should be useful for providing a heavy-atom derivative for crystals of enzymes that bind ATP.     

Inhibition by active site directed covalent modification of human glyoxalase I

The glyoxalase pathway is responsible for conversion of cytotoxic methylglyoxal (MG) to d-lactate. MG toxicity arises from its ability to form advanced glycation end products (AGEs) on proteins, lipids and DNA. Studies have shown that inhibitors of glyoxalase I (GLO1), the first enzyme of this pathway, have chemotherapeutic effects both in vitro and in vivo, presumably by increasing intracellular MG concentrations leading to apoptosis and cell death. Here, we present the first molecular inhibitor, 4-bromoacetoxy-1-(S-glutathionyl)-acetoxy butane (4BAB), able to covalently bind to the free sulfhydryl group of Cys60 in the hydrophobic binding pocket adjacent to the enzyme active site and partially inactivate the enzyme. Our data suggests that partial inactivation of homodimeric GLO1 is due to the modification at only one of the enzymatic active sites. Although this molecule may have limited use pharmacologically, it may serve as an important template for the development of new GLO1 inhibitors that may combine this strategy with ones already reported for high affinity GLO1 inhibitors, potentially improving potency and specificity.     

Pyruvate carboxylase: affinity labelling of the magnesium adenosine triphosphate binding site.

The 2' , 3'-dialdehyde derivative of ATP (oATP) was prepared by periodate oxidation and on the following criteria was considered to be an effective affinity label. The magnesium complex of this derivative (Mg-oATP2) was shown to ba linear competitive inhibitor with respect to MgATP2-in both the acetyl-CoA-dependent and -independent activities of the enzyme but was a non-competitive inhibitor with respect to bicarbonate, and an uncompetitive inhibitor with respect to pyruvate. Mg-oATP was covalently bound to pyruvate carboxylase by reduction using sodium borohydride with concurrent irreversible inactivation of the enzyme...     

Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine.

DNA repair methyltransferases (MTases) remove methyl or other alkyl groups from the O6 position of guanine or the O4 position of thymine by transfering the group to an active site cysteine. In order to trap an MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-aminopurine (d6Cys2AP) was synthesized and incorporated into oligonucleotides. d6Cys2AP has a disulfide bond within an alkyl chain linked to the 6 position of 2,6-diaminopurine, which disulfide can be reduced to form a free thiol. Addition of human MTase to reduced oligonucleotide resulted in a protein-DNA complex that was insensitive to denaturation by SDS and high salt, but which readily dissociated in the presence of dithiothreitol. Formation of this complex was prevented by methylation of the active site cysteine. Evidence that the active site cysteine is directly involved in disulfide bond formation was obtained by N-terminal sequencing of peptides that remained associated with DNA after proteolysis of the complex.     

Identification of subsidiary catalytic groups at the active site of beta-ketoacyl-CoA thiolase by covalent modification of the protein

beta-Ketoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) is known to possess sulfhydryl groups of cysteines at the active site that are essential for its catalytic activity. Other groups at the active site that participate in the catalytic process were identified by using anhydride reagents which covalently modify the protein by specifically reacting with any amino groups potentially present at the active site. Since these reagents may also react with thiol groups, the enzyme's amino groups were modified after masking the cysteine thiols present by an alkylalkane thiosulfonate-type reagent, methyl methanethiol-sulfonate (MMTS), that selectively formed a disulfide bridge, thus generating an inactive thiolmethylated enzyme. When this procedure was followed, the enzyme could be undoubtedly modified at its amino by the anhydride reagent, leading to a doubly modified protein. The thiomethyl group could then be removed by reduction with dithiothreitol, yielding an enzyme modified solely on the amino residues. The amino group could be unblocked in turn by exposure to acidic pH. The different anhydrides inactivated thiolase, but only acetoacetyl coenzyme A (AcAcCoA) provided any protection against inactivation. When thiolmethylcitraconyl thiolase was reduced with dithiothreitol the enzyme remained inactive, but when the doubly modified enzyme was exposed to pH 5 then the reduction led to formation of an active enzyme. These results are interpreted as demonstrating a role for an amino group at the enzyme active site. A catalytic mechanism is proposed for the enzyme which involves the amino group.     

Probes of catalytic site cooperativity during catalysis by the chloroplast adenosine triphosphate and the adenosine triphosphate synthase

During net nucleoside triphosphate synthesis by chloroplast ATP synthase the extent of water oxygen incorporation into each nucleoside triphosphate released increases with decrease in ADP, GDP or IDP concentration. Likewise, during net ATP hydrolysis by the Mg2+-activated chloroplast ATPase, the extent of water oxygen incorporation into each Pi released increases as the ATP, GTP, or ITP concentration is decreased. However, the concentration ranges in which substrate modulation occurs differs with each nucleotide. Modulation of oxygen exchange during synthesis and hydrolysis of adenine nucleotides, as measured by variation in the extent of water oxygen incorporation into products, occurs below 250 microM. In contrast, guanosine and inosine nucleotides alter the extent of exchange at higher and much wider concentration ranges. Activation of the chloroplast ATPase by either heat or trypsin results in similar catalytic behavior as monitored by ATP modulation of oxygen exchanges during hydrolysis in the presence of Mg2+. More exchange capacity is evident with octylglucoside-activated enzyme at all ATP concentrations. High levels of tentoxin were also found to alter the catalytic exchange parameters resulting in continued water oxygen exchange into Pi released during hydrolysis at high ATP concentrations. Little or no oxygen exchange accompanies ATP hydrolysis in the presence of Ca2+. The [18O]Pi species formed from highly gamma-18O-labeled ATP at lower ATP concentrations gives a distribution as expected if only one catalytic pathway is operative at a given ATP concentration. This and other results support the concept of catalytic cooperativity between alternating sites as explanation for the modulation of oxygen exchange by nucleotide concentration.     

Dibutylchloromethyltin chloride, a covalent inhibitor of the adenosine triphosphate synthase complex

1. The synthesis of dibutylchloromethyltin chloride, a new covalent inhibitor of the mitochondrial ATP synthase [oligomycin-sensitive ATPase (adenosine triphosphatase)] complex is described, together with a method for preparing dibutylchloro[(3)H]methyltin chloride. 2. Studies with the yeast mitochondrial oligomycin-sensitive ATPase complex show that dibutylchloromethyltin chloride inhibits both the membrane-bound enzyme and also the purified Triton X-100-dispersed preparation. 3. F(1)-ATPase is not inhibited even at 500nmol of dibutylchloromethyltin chloride/mg of protein, and the general inhibitory properties are similar to those of triethyltin, oligomycin and dicyclohexylcarbodi-imide, known energy-transfer inhibitors of oxidative phosphorylation. 4. Binding studies with yeast submitochondrial particles show that dibutylchloromethyltin chloride antagonizes the binding of triethyl[(113)Sn]tin, indicating that there is an interaction between the two inhibitor-binding sites. 5. Unlike triethyltin, inhibition by dibutylchloromethyltin chloride is due to a covalent interaction which titrates a component of the inner mitochondrial membrane present at a concentration of 8-9nmol/mg of protein. 6. All of the labelled component can be extracted with chloroform/methanol (2:1, v/v), and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chloroform/methanol extract indicates that the labelled component has an apparent mol.wt. of 6000-8000. However, t.l.c. reveals the presence of only one labelled component which is lipophilic and non-protein and is distinct from the free inhibitor, mitochondrial phospholipids and the dicyclohexylcarbodi-imide-binding protein (subunit 9). 7. Inhibition of mitochondrial ATPase and oxidative phosphorylation is correlated with specific interaction with a non-protein lipophilic component of the mitochondrial inner membrane which is proposed to be a co-factor or intermediate of oxidative phosphorylation.     

NMR of a synthetic peptide spanning the triphosphate binding site of adenosine 5'-triphosphate in actin

The amino acid residues 114-118 in actin were found to be implicated strongly in the binding of nucleotide, and as would be expected for such an important binding site, they are located in a completely conserved region of the actin sequence. A 19-residue peptide with the actin sequence 106-124 was synthesized in order to span the putative triphosphate binding site. Proton NMR spectra of the actin peptide 114-118 in the presence and absence of ATP indicated that Arg-116 and Lys-118 are particularly involved in binding ATP. A strong binding of ATP to the peptide 106-124 also was measured. Tripolyphosphate bound to the peptide 106-124 somewhat more weakly than ATP. Binding involved residues 115-118 and 121-124, indicating the presence of a reverse turn between these segments. Proton resonances were assigned by using two-dimensional double quantum correlated spectroscopy, one-dimensional spin decoupling techniques, one-dimensional nuclear Overhauser enhancement difference spectroscopy, and pH titration. The alpha CH resonances of Ala-3 and Asn-6 are markedly shifted downfield with respect to values in small unstructured peptides due to their close proximity to the side chains of Pro-4 and Pro-7, respectively. Several other resonances display chemical shifts which are indicative of a structured environment. Assignment of the amide proton resonances in H2O and measurements of the coupling constant 3JHNCH and the chemical shifts of the amide protons reveal that much of the synthetic peptide, particularly the backbone, exhibits a highly structured environment and represents a good model for the triphosphate binding site in actin.     

Action of the adenosine triphosphate analog, adenylyl imidodiphosphate in mitochondria.

Adenylyl imidodiphosphate (AMP-PNP), and analog of adenosine triphosphate (ATP), is a potent competitive inhibitor of mitochondrial ATPase activity. It inhibits both the soluble oligomycin-insensitive ATPase (K_i = 9.2 × 10^?7 M) and the bound oligomycin-sensitive APTase (K_i = 1.3 × 10^?6 M). ATPase activity of inside-out submitochondrial preparations are more sensitive to AMP-PNP in the presence of an uncoupler (K_i = 2.0 × 10^?7 M). Mitochondrial ATP-dependent reactions (reversed electron transfer and potassium uptake) do not proceed if ATP is replaced with AMP-PNP; however, the analog does affect these systems. Oxidative phosphorylation of whole mitochondria and submitochondrial preparations were unaffected by AMP-PNP.     

pH dependence of the effect of adenosine triphosphate and ethylenediaminetetraacetate on sodium and magnesium binding by cellular membrane fragments

The pH dependence of previously reported effects of adenosine triphosphate (ATP) and ethylenediaminetetraacetate (EDTA) on cation binding by rat liver microsomes was studied by an equilibration and washing procedure. Equilibration of microsomes in media containing 95 mM NaCl and 4 mM MgCl₂ with pH varied from 4 to 8 resulted in an increase in bound cations from zero below pH 4 to 0.90 mmoles Mg and 0.34 mmoles Na/g N at pH 8; the ratio of bound Na/bound Mg increased from 0.15 at pH 5 to 0.38 at pH 8. Addition of 5 mM EDTA to the equilibration media produced striking changes in cation binding such that bound Na/bound Mg increased from 0.30 at pH 5 to 3.90 at pH 7 and decreased to 3.55 at pH 8. In the presence of added 10 mM ATP, bound Na/bound Mg increased from 0.10 at pH 5 to a maximum of 0.80 at pH 7. The observed changes could generally be correlated with known mass law relationships, although the system containing added ATP was complicated considerably by the hydrolysis of ATP. Results demonstrate that environmental pH is an important factor in determining the effect of ATP and EDTA on the cation binding pattern of cellular membranes. Because hydrogen ion is a product of ATP hydrolysis as well as of other metabolic reactions, the described interactions may be of particular significance in the molecular mechanisms of ATP effects on cation binding and transport in living cells.     

Reaction of the histidines of prolactin with ethoxyformic anhydride. A binding site modification.

The seven histidines of bovine prolactin were modified with ethoxyformic anhydride and two classes of reactivity were apparent: 5 histidines were in the more reactive class (k = 0.097 min-1) and 2 histidines were less reactive (k = 0.011 min-1). The activity of the modified prolactins was determined by measuring their ability to bind to prolactin receptors from rabbit mammary glands. This assay showed that prolactin was fully active when 0 to 5 histidines were modified. If all 7 residues were modified, the hormone was unable to bind to its receptor. Circular dichroism studies indicated no significant differences in conformation for prolactins which had 2 to 7 histidines modified. Modification of human growth hormone and human placental lactogen with ethoxyformic anhydride resulted in a loss of the ability of these lactogenic hormones to bind to the prolactin receptor. For all three hormones, essentially full activity was recovered when the modifying group was removed by treatment with hydroxylamine. Sequence comparisons indicate that only 2 of the 3 growth hormone histidines and 2 of 7 placental lactogen histidines were homologous with histidines in bovine prolactin and that, in each case, they correspond to His-27 and His-30 in bovine prolactin. It is postulated that these residues serve to identify a portion of the binding domain of bovine prolactin.     

Design of new inhibitors specific to the adenosine triphosphate binding site of DNA topoisomerases II

Summary DNA topoisomerases II are involved in the segregation of chromosomes which occurs after DNA replication. These enzymes proceed by nicking and resealing of a phosphodiester bond of the DNA double helix and require the hydrolysis of ATP into ADP and inorganic phosphate. Studies of ATP hydrolysis showed specific properties according to the source of isolation of the enzymes, suggesting the existence of an evolution of the ATP binding site of DNA topoisomerases II. In order to study this evolution, two experimental strategies were followed, first of all an analysis of the topography of the ATP binding site by forming UV crosslinks between ATP and the enzymes, and second the effects of new inhibitors.