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.     

Proximate sulfhydryl groups in the acetylglutamate complex of rat carbamylphosphate synthetase I: their reaction with the affinity reagent 5'-p-fluorosulfonylbenzoyladenosine

A preparation of rat carbamylphosphate synthetase I, isolated in the presence of antipain and stable without glycerol, has been used to investigate the effect of the allosteric activator, N-acetyl-L-glutamate (AcGlu), on the sulfhydryl chemistry of the enzyme. The enzyme X AcGlu complex was rapidly inactivated by several sulfhydryl group reagents and the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSO2BzAdo), with the loss of two sulfhydryl groups per monomer. Inactivation was much slower without AcGlu, and ATP/Mg2+/K+ provided complete protection. Reaction with a 1.1 molar excess of 4,4'-dipyridyldisulfide resulted in an intramonomer disulfide bond between groups that are probably juxtaposed in the activated enzyme, because 1.1 equivalents of the vicinal dithiol reagent, phenylarsine oxide, eliminated the rapid reaction with the disulfide. Evidence is presented that the same disulfide bond was formed in the reactions with 5-thiocyano-2-nitrobenzoic acid and FSO2BzAdo. Inactivation by FSO2BzAdo was a pseudo-first-order reaction. The concentration dependence of the rate is consistent with the reaction proceeding through a noncovalent complex (KI = 67 microM and k2 = 0.23 min-1 at pH 7.0, 30 degrees C). Protection from FSO2BzAdo by ATP required Mg2+ in excess of ATP with KMgATP = 4.5 microM at saturating free Mg2+ (0.1 M K+) and KMg2+ = 6.5 mM. KMgATP is close to Kd for the molecule of ATP that contributes the phosphoryl group of carbamylphosphate (H.B. Britton, V. Rubio, and S. Grisolia, (1979) Eur. J. Biochem. 102, 521-530]; KMg2+ agrees with the minimum value for the steady-state kinetic parameter, Ki,Mg2+, obtained under the same conditions. Dissociation constants for adenosine (320 microM), MgADP (110 microM) at 10 mM Mg2+, and AcGlu (100 microM) were also estimated.     

Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine

The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied. This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity. Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center. When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics. The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment. Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R. B., Rhee, S. G., Luterman, D. L., and Chock, P. B. (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme. The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity. The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.     

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase.

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.     

Phosphate binding and ATP-binding sites coexist in Na+/K(+)-transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4.

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K(+)-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): Co(NH3)4PO4 + E2 Kd in equilibrium E2.Co(NH3)4PO4k2----E'2.Co(NH3)4PO4. The inactivation rate constant k2 for the formation of a stable E'2.Co(NH3)4PO4 at 37 degrees C was 0.057 min-1; the dissociation constant, Kd = 300 microM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta, gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks = 50 microM, as did phosphate (Ks = 16 mM) and Mg2+ (Ks = 160 microM). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (Ks = 860 microM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 microM, and for the E.Co(NH3)4PO4 complex of 720 microM, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K(+)-ATPase and K(+)-activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na(+)-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P-labelled tetramminecobalt ATP ([gamma-32P]Co(NH3)4ATP) at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K(+)-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(III) ATP ([gamma-32P]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K(+)-ATPase by [gamma-32P]CrATP prevented the binding of Co(NH3)4 32PO4 or [gamma-32P]Co(NH3)4ATP to the enzyme. [gamma-32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha,beta)2-diprotomer, which change their interactions during the Na+/K(+)-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.     

Affinity labeling of cAMP-dependent protein kinase with p-fluorosulfonylbenzoyl adenosine. Covalent modification of lysine 71

p-Fluorosulfonylbenzoyl 5'-adenosine (FSO2BzAdo) was shown previously to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase II from porcine skeletal muscle (Zoller, M. J., and Taylor, S. S. (1979) J. Biol. Chem. 254, 8363-8368). The catalytic subunit of porcine heart cAMP-dependent protein kinase was also inhibited following incubation with FSO2[14C]BzAdo, and inhibition was shown to result from the stoichiometric, covalent modification of a single lysine residue. The amino acid sequence in an extended region around the carboxybenzenesulfonyl lysine (CBS-lysine) was elucidated by characterizing both tryptic and cyanogen bromide peptides containing the 14C-modified residue. The sequence in this region was Leu-Val-Lys-His-Lys-Glu-Thr-Gly-Asn-His-Phe-Ala-Met-Lys(CBS)-Ile-Leu-Asp-Lys-Glu-Lys-Val-Val-Lys-Leu-Lys-Gln-Ile. The covalently modified residue corresponded to lysine 71 in the overall polypeptide chain. Homologies to bovine heart catalytic subunit and to a site modified by FSO2BzAdo in phosphofructokinase are considered.     

The inhibition of phosphatidylinositol 3-kinase by quercetin and analogs.

Phosphatidylinositol (PtdIns) 3-kinase is an enzyme involved in cellular responses to growth factors. Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyrano-4-one), a naturally occuring bioflavinoid, was found to inhibit PtdIns 3-kinase with an IC50 of 1.3 micrograms/ml (3.8 microM); inhibition appears to be directed towards the ATP binding site of the kinase. Analogs of quercetin were also investigated as PtdIns 3-kinase inhibitors, with the most potent compounds exhibiting IC50's in the range of 1.7-8.4 micrograms/ml (5-19 microM). In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 greater than 30 micrograms/ml). These findings suggest that flavinoids may serve as potent inhibitors of PtdIns 3-kinase. Furthermore, the enzyme is much more sensitive to substituents at the 3-position of the flavinoid ring than are other protein and PtdIns kinases, suggesting that specific inhibitors of PtdIns 3-kinase can be developed to explore the biological role of the enzyme in cellular proliferation and growth factor response.     

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.     

Effect of modification of lysine residues of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase with pyridoxal 5'-phosphate

Inactivation of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase by pyridoxal 5'-P followed by reduction with NaBH4 was studied. Fructose-6-P,2-kinase is over 80% inactivated by 2 mM pyridoxal 5'-P. The stoichiometry of the pyridoxyl-P incorporation and the inactivation of the kinase follows a biphasic curve. The first P-pyridoxyl residue incorporated per protomer does not affect fructose-6-P,2-kinase, but the next two P-pyridoxyl incorporation/protomer results in 80% inactivation. The Km values for ATP and fructose-6-P of the enzymes containing varying amounts of P-pyridoxyl groups at intermediate levels of inactivation are not altered, but Vmax is decreased. Among the metabolites tested, only fructose-2,6-P2 and Mg-ATP are competitive with pyridoxal-P and protect the enzyme against the inactivation. Neither the activity nor the fructose-6-P inhibition of fructose-2,6-bisphosphatase is affected by the modification. The acid hydrolysate of the inactive P-[3H]pyridoxyl enzyme contained only [3H]pyridoxyl lysine. High performance liquid chromatography of tryptic peptides of phospho[3H]pyridoxyl enzymes reveals two peptides which were missing in the enzyme protected by fructose-2,6-P2 or ATP during the modification reaction. These peptides have been isolated, and their amino acid sequences have been determined as Asp-Gln-Asp-Lys-Tyr-Arg and Asp-Val-His-Lys-Tyr. Pyridoxal-P reacts specifically with two lysine residues at the fructose-2,6-P2-binding site of fructose-6-P,2-kinase but not that of fructose-2,6-bisphosphatase. The site may also overlap with the ATP-binding site.     

Rhodopsin kinase: substrate specificity and factors that influence activity

Rhodopsin kinase was prepared from bovine retinas by the method of Sitaramayya [Sitaramayya, A. (1986) Biochemistry 25, 5460] with some minor modifications. The enzyme is able to phosphorylate bovine rhodopsin in the disk membrane, rhodopsin from other species, and rhodopsin solubilized in mild detergent (dodecyl maltoside). Rhodopsin kinase can phosphorylate synthetic peptides containing the appropriate sequences from bovine rhodopsin; however, the Km values for these peptides are about 3 orders of magnitude higher than that for rhodopsin or ATP. Some peptides from the cytosolic surface of rhodopsin inhibit the phosphorylation. These results suggest that more than one region of rhodopsin is involved in the interaction of rhodopsin of the kinase. Mg2+ is required for the Mg-ATP complex as shown by the observation that (ethylenedinitrilo)tetraacetic acid inhibits kinase activity. Second, free Mg2+ above the concentration required to complex all of the ATP present activates the kinase. Third, higher concentrations of Mg2+ yield Mg-ATP-Mg instead of Mg-ATP and therefore inhibit the kinase activity. Other physiologically important cations such as Ca2+, Na+, and K+ reduce the activity of the kinase, probably by forming a metal ion-ATP complex, thereby reducing the concentration of Mg-ATP. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSO2BzAdo), an inhibitor of kinases and ATPases, inhibits rhodopsin kinase according to pseudo-first-order kinetics. The relationship between the first-order constant and the concentration of FSO2BzAdo is hyperbolic. This indicates that a reversible complex between the ATP analogue and the enzyme is formed prior to the covalent attachment of the analogue to rhodopsin kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ATP-binding site of brain phosphatidylinositol 4-kinase PI4K230 as revealed by 5'-p-fluorosulfonylbenzoyladenosine

The ATP-binding site of purified bovine brain phosphatidylinositol 4-kinase 230 (PI4K230) was studied by its reaction with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an ATP-like alkylating reagent. Four hundred to eight hundred micromolar FSBA inactivated PI4K230 specifically with apparently first-order kinetics and resulted in 50% loss of enzyme activity in 36--130 min. The specificity of the reaction with FSBA was demonstrated by the lack of inactivation with 5'-p-fluorosulfonylbenzoyl chloride and by protection with ATP and ATP analogues against inactivation. Most ATP analogues competed with FSBA inactivation in order of their increasing hydrophobicity, parallel to their inhibitory potency in activity measurements. The specific binding of FSBA to PI4K230 was demonstrated also by Western-blot experiments. These results suggest that FSBA-reactive group(s) involved in the enzyme activity are located near to the ATP-binding site in a hydrophobic region of native PI4K230. Experiments with site-directed mutagenesis indicate that the conserved Lys-1792 plays essential role in the enzyme activity and serves as one target of affinity labelling by FSBA. Prevention of both Lys-1792-directed and Lys-1792-independent binding of FSBA by Cibacron Blue 3GA suggest that these sites are located spatially close to each other.     

ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm)

ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.     

Affinity modification of creatine kinase from rabbit skeletal muscle by 2',3'-dialdehyde derivatives of ADP and ATP

Periodate-oxidized ADP and ATP (oADP and oATP) are substrates and affinity reagents for creatine kinase from rabbit skeletal muscle. oADP and oATP modified a lysine epsilon-amino group in the nucleotide-binding site of the enzyme. Complete inactivation is observed upon binding 2 moles oADP per 1 mole of the enzyme dimer. Modification with oADP is described by a liner dependence of the log of enzyme activity on time, testifying to a pseudo-first-order of the reaction. The reaction rate constant (ki = 8.10(3) min-1) and dissociation constant for the reversible enzyme-oADP complex (Kd = 62 microM) were determined. ADP protected the enzyme from inactivation and covalent binding of the analog, whereas oADP covalently bound to the enzyme was phosphorylated by phosphocreatine. The data obtained allow to suggest that the epsilon-amino group of a lysine residue of the active site is located in close proximity to ribose of ATP and ADP forming a complex with the enzyme. This group seems essential for correct orientation of the nucleotide polyphosphate chain in the enzyme active center, but take no immediate part in the transphosphorylation process.     

How do MgATP analogues differentially modify high-affinity and low-affinity ATP binding sites of Na+/K(+)-ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.     

Inhibition of adenylate cyclase by the 2',3'-dialdehyde of adenosine triphosphate

The periodate-oxidized analog of ATP, 2',3'-dialATP, competitively inhibited bovine brain and rat liver adenylate cyclase. The apparent Ki for inhibition of brain adenylate cyclase by 2',3'-dialATP was 196 microM in the presence of Mg2+ and 37 microM in the presence of Mn2+. The Ki values for inhibition of rat liver adenylate cyclase by 2',3'-dialATP were 48 and 30 microM in the presence of Mg2+; and Mn2+, respectively. Adenylate cyclase activity was irreversibly inactivated by 2'3'-dialATP in the presence of NaCNBH3 and the kinetics for loss in enzyme activity were pseudo-first order. Both ATP and Tris protected adenylate cyclase from irreversible inhibition by 2',3'-dialATP and NaCNBH3. It is proposed that 2',3'-dialATP forms a Schiff's base with an amino group at the active site of the enzyme and that Na-CNBH3 reduction of this Schiff's base causes irreversible modification of the catalytic subunit. The Km for 2',3'-dialATP inactivation, the maximal rate constant of inactivation, and protection of the enzyme by ATP were not affected by the presence or absence of free Mg2+. These data indicate that a divalent cation is not required for binding of 2',3'-dialATP to the active site of adenylate cyclase.     

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.     

Evidence for a Mg2+-induced conformational change at the ATP-binding site of (Na+ + K+)-ATPase demonstrated with a photoreactive ATP-analogue

1. The 3'-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid has been prepared and its ability to act as a photoaffinity label of (Na+ + K+)-ATPase has been tested. 2. In the dark 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]adenosine triphosphate (N3-ATP) is a substrate of (Na+ + K+)-ATPase and a competitive inhibitor of ATP hydrolysis. 3. Upon irradiation by ultraviolet light, N3-ATP photolabels the high-affinity ATP-binding site and is covalently attached to the alpha-subunit and an approximately 12000-Mr component. 4. Photolabeling of the alpha-subunit by N3-ATP irreversibly inactivates (Na+ + K+)-ATPase. 5. Photoinactivation is strictly Mg2+-dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation. 6. Mg2+, in concentrations required for photoinactivation, protects (Na+ + K+)-ATPase against inactivation by tryptic digestion under controlled conditions. 7. It is assumed that a conformational change of the ATP-binding site of (Na+ + K+)-ATPase occurs upon binding of Mg2+ to a low-affinity site.     

Affinity labeling of spinach leaf phosphoribulokinase by ATP analogs. Modification of an active site lysine

Spinach leaf phosphoribulokinase is sensitive to modification by ATP analogs that react with lysine residues. The 2',3'-dialdehyde derivative of ATP (oATP) inactivates enzyme in a slow, time-dependent fashion. The process follows first-order kinetics (kinact = 0.07 min-1), and the concentration dependence of inactivation indicates tight inhibitor binding (Ki = 106 microM). ATP offers good protection against inactivation (Kd = 67 microM), suggesting that oATP is directed toward the catalytic site. This conclusion is supported by the fact that oATP functions as an alternate substrate (Km = 0.55 mM). Inactivation of phosphoribulokinase by [14C]oATP results in a modification stoichiometry of 0.7/site. The 14C-labeled enzyme is stable to dialysis, suggesting that the covalent adduct formed between protein and oATP is not a simple Schiff's base. Adenosine di- and triphosphopyridoxals (Ado-P2-Pl and Ado-P3-Pl, respectively) also inhibit spinach phosphoribulokinase in a time-dependent fashion. In this case, activity loss is reversible unless the inhibited species is borohydride-reduced, suggesting that Ado-P2-Pl and Ado-P3-Pl form Schiff's bases with an amino group on the enzyme. Protection is afforded by the substrate ATP, suggesting that modification is active site-directed. Prolonged incubation of enzyme with these inhibitors does not result in complete inactivation of phosphoribulokinase. Residual activity is dependent on inhibitor concentration, as would be expected if equilibrium is established between the noncovalent E.I complex and the covalent (Schiff's base) E-I species. Kinetic data analysis indicates Ki values of 175 and 11 microM for Ado-P2-Pl and Ado-P3-Pl, respectively. Thus, the ATP-binding domain can easily accommodate the pyridoxal moiety which is tethered to the polyphosphate chain. The phosphorylated ATP analogs employed in this study exhibit substantially tighter binding to phosphoribulokinase than does fluorosulfonyl-benzoyladenosine (Ki = 4.8 mM), which we have previously demonstrated to be useful in selectively modifying the ATP-binding domain (Krieger, T. J., and Miziorko, H. M. (1986) Biochemistry 25, 3496-3501; Krieger, T. J., Mende-Mueller, L. M., and Miziorko, H. M. (1987) Biochim. Biophys. Acta 915, 112-119). Although the adduct formed between oATP and enzyme was unsuitable for structural analysis, borohydride reduction of the Schiff's base formed between enzyme and Ado-P3-[3H]Pl produced a species useful for investigation by protein chemistry techniques. A radiolabeled tryptic peptide was prepared, isolated, and sequenced; the data indicate that lysine 68 is the residue modified by Ado-P3-[3H]Pl.     

Dual functional roles of ATP in the human mitochondrial malic enzyme

Human mitochondrial malic enzyme is a regulatory enzyme with ATP as an inhibitor. Structural studies reveal that the enzyme has two ATP-binding sites, one at the NAD(+)-binding site in the active center and the other at the exo site in the tetramer interface. Inhibition of the enzyme activity is due to the competition between ATP and NAD(+) for the nucleotide-binding site at the active center with an inhibition constant of 81 microM. Binding of the ATP molecule at the exo site, on the other hand, is important for the maintenance of the quaternary structural integrity. The enzyme exists in solution at neutral pH and at equilibrium of the dimer and tetramer with a dissociation constant (K(TD)) of 0.67 microM. ATP, at a physiological concentration, shifts the equilibrium toward tetramer and decreases the K(TD) by many orders of magnitude. Mutation of a single residue Arg542 at the tetrameric interfacial exo site resulted in dimeric mutants. ATP thus has dual functional roles in the mitochondrial malic enzyme.     

The stereoselective recognition of substrates by phosphoinositide kinases. Studies using synthetic stereoisomers of dipalmitoyl phosphatidylinositol.

Soluble phosphatidylinositol (PtdIns) 4- and 3-kinase activities were partially purified and characterized from human placental extracts. The placental PtdIns 4-kinase (type 3) has a Km for ATP of 460 microM and is kinetically different to a partially purified human erythrocyte, membrane-bound, PtdIns 4-kinase (type 2). These three inositol lipid kinases were then used to compare their substrate specificities against the four synthetic stereoisomers of dipalmitoyl PtdIns. Only the placental 4-kinase was influenced by the chirality of the glycerol moiety of PtdIns. However, neither of the 4-kinases was able to phosphorylate L-PtdIns and, therefore, these kinases have an absolute requirement for the inositol ring to be linked to the glyceryl backbone of the lipid through the D-1 position. Phosphoinositide 3-kinase, on the other hand, was found to phosphorylate both D- and L-PtdIns. While the 3-kinase phosphorylated exclusively the D-3 position of D-PtdIns, further analyses demonstrated that the same enzyme phosphorylated two sites on L-PtdIns, namely the D-6 and D-5 positions of the inositol ring. Some implications of these findings are discussed.