Identification of a tyrosine residue at a nucleotide binding site in the beta subunit of the mitochondrial ATPase with p-fluorosulfonyl[14C]-benzoyl-5'-adenosine.

The mitochondrial F1-ATPase is irreversibly inactivated by the adenine nucleotide analogue, p-fluorosulfonylbenzoyl-5'-adenosine. This inactivation is partly prevented by the presence of bound adenine nucleotides. Inactivations of the ATPase with p-fluorosulfonyl[14C]benzoyl-5'-adenosine were most efficiently accomplished with the nucleotide-free enzyme at pH 7.0, in a buffer containing 20% glycerol. Under these conditions, 4.2 g atoms of 14C are incorporated per 350,000 g of enzyme when the ATPase is inactivated by 90% by its reaction with 2 mM p-fluorosulfonyl[14C]benzoyl-5'-adenosine. Isolation of the component polypeptide chains of the labeled ATPase showed that all of the radioactivity was associated with the two largest subunits. The isolated alpha subunit contained 0.45 g atom of 14C/mol and the isolated beta subunit contained 0.88 g atom of 14C/mol. Hence, the inactivation can be correlated with the incorporation of 14C into the beta subunit. This suggests that the hydrolytic site of the enzyme resides on this subunit. The majority of the radioactivity in a tryptic digest of labeled beta subunit is contained ina tryptic peptide that has the following amino acid sequence: Ile-Met-Asp-Pro-Asn-Ile-Val-Gly-Ser-Glu-His-Tyr-Asp-Val-Ala-Arg, where Tyr is the radioactive derivative of the tyrosine residue that was sulfonylated during the inactivation.     

Periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate as a fluorescent affinity label for pigeon liver malic enzyme

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (KI) and the maximum inactivation rate constant at saturating reagent concentration (kmax) were found to be 1.1 +/- 0.02 mM and 0.068 +/- 0.001 min-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the KI value whereas high malate concentration increased the kmax. Mn2+ or NADP+ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP+ or Mn2+. Maximum and synergistic protection was afforded by NADP+, Mn2+ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP+ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a Ki value of 4.1 +/- 0.1 microM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)+-dependent dehydrogenases.     

Escherichia coli F1-ATPase can use GTP-nonchaseable bound adenine nucleotide to synthesize ATP in dimethyl sulfoxide.

Escherichia coli F1-ATPase contained 3 mol of tightly-bound adenine nucleotide/mol enzyme. A further 3 mol could be loaded by incubation of the enzyme with ATP. The unloaded enzyme was designated as a F1[2,1] type on the basis of the ability of GTP to displace 1 mol of adenine nucleotide/mol of F1 [Kironde, F.A.S., & Cross, R.L. (1986) J. Biol. Chem. 261, 12544-12549]. The loaded enzyme was designated F1[3,3] since GTP could displace 3 of the 6 mol of bound adenine nucleotide/mol of F1. Incubation of F1[2,1], F1[2,0], and F1[3,0] with phosphate in the presence of 30% (v/v) dimethyl sulfoxide led to the synthesis of ATP from endogenous bound ADP. Hydrolysis of newly synthesized ATP occurred on transfer of the F1 from 30% (v/v) dimethyl sulfoxide to an entirely aqueous medium. Thus, synthesis and hydrolysis of ATP can occur at GTP-nonchaseable adenine nucleotide binding sites, and these sites in dimethyl sulfoxide are not necessarily equivalent to noncatalytic sites.     

Synthesis of a reactive ATP analog and its preliminary application as an affinity labeling reagent

A reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP.PCP, was synthesized in an attempt to covalently label the binding sites for adenine nucleotides, especially ATP, of various enzymes which utilize adenine nucleotides as substrates, cofactors, inhibitors or allosteric effectors. This reagent rapidly inactivated rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD), myokinase (MK), and creatine kinase (CK) under very mild conditions. Adenine nucleotide substrates prevented the inactivation. In the case of GPD, complete inactivation was observed when 1 mol of the reagent per mol of enzyme subunit was incorporated into the enzyme. These results indicate that the present ATP analog may be useful as an affinity labeling reagent for various adenine nucleotide-dependent enzymes.     

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.     

Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase

The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked inhibition.     

Covalent modification of a highly reactive and essential lysine residue of ribulose-1,5-bisphosphate carboxylase/oxygenase activase.

Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. Incubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-1 s-1 compared to 21 M-1 s-1 for sulfo-NHS-acetate. Inactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mol of AMCA per mol of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acid-lysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of K247 accounted for 1 mol of AMCA incorporated per mol of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS- and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the gamma-phosphate showed that K247 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of K247 is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.     

Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3'-O-(4-benzoyl)benzoyl ADP

3'-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF1). As with the CF1-ATPase (Bar-Zvi, D. and Shavit, N. (1984) Biochim. Biophys. Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF1-ATPase. BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CF1-ATPase, it has no effect on the Vmax. In the absence of Mg2+ 1 mol BzADP binds noncovalently to TF1, while with Mg2+ 3 mol are bound. Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF1 and correlates with the inactivation of the ATPase. Complete inactivation of the TF1-ATPase occurs after covalent binding of 2 mol BzADP/mol TF1. Photoinactivation of TF1 by BzADP is prevented if excess of either ADP or ATP is present during irradiation. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bz[3H]ADP-labeled TF1-ATPase shows that all the radioactivity is incorporated into the beta subunit.     

The interaction of N,N'-dicyclohexylcarbodiimide with chloroplast coupling factor 1

1. Incubation of soluble spinach Coupling Factor 1 (CF1) with dicyclohexylcarbodiimide (DCCD) results in the inactivation of the ATPase. The DCCD inactivation is time- and concentration-dependent. Complete inactivation of the CF1-ATPase activity requires the binding of 2 mol of DCCD/mol of CF1. The binding sites of DCCD are located on the beta subunit of CF1. 2. DCCD modification of soluble CF1 eliminates one adenine nucleotide binding site which is exposed by dithiothreitol activation or by incubation with tentoxin. The inactivation of both the ATPase activity and the adenine nucleotide binding site are pH-dependent. The inactivation of both the ATPase activity and the adenine nucleotide binding site are pH-dependent. Half-maximal inhibition occurs at about pH 7.5. 3. The DCCD-modified CF1, reconstituted with EDTA-treated chloroplasts, is fully active is restoring proton uptake but not in restoring ATP synthesis or light-dependent adenine nucleotide exchange.     

Species-specific irreversible inhibition of Neisseria gonorrhoeae dihydrofolate reductase by a substituted 2,4-diamino-5-benzylpyrimidine

Neisseria gonorrhoeae dihydrofolate reductase undergoes a time-dependent, irreversible inactivation by 2,4-diamino-5-[3,5-dimethoxy-4-(p-bromoacetamidophenoxy)benzyl] pyrimidine. The kinetics of inactivation are consistent with the reversible formation of an enzyme-inhibitor complex followed by covalent binding to the enzyme. The reversible component is competitive with dihydrofolate and has an inhibitor binding constant of 10 nM. Irreversible inactivation proceeds as a pseudo first-order process with a minimum inactivation half-time of 20 min and a Ki of 28 nM. Using radiolabeled inhibitor, it was shown that approximately 1 mol of ligand was covalently bound to the enzyme/mol of methotrexate binding site when the enzyme was completely inhibited. Radiolabeled inhibitor remained associated with the enzyme following denaturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide cleavage of the 14C-labeled enzyme-inhibitor complex yielded only one radioactive polypeptide, and sequence determinations showed that His-25 was modified by covalent attachment of the inhibitor. When dihydrofolate reductases from Lactobacillus casei, Streptococcus faecium, Escherichia coli, SR-1 rodent lymphoma, and chicken liver were tested with the affinity label, only the L. casei enzyme showed a time-dependent increase in inhibition. These data, along with comparisons of known amino acid sequences and x-ray crystal structures, were used to make predictions concerning the three-dimensional conformation of the gonococcal enzyme.     

Affinity labeling of adenine nucleotide-related enzymes with reactive adenine nucleotide analogs. I. Affinity labeling of glyceraldehyde 3-phosphate dehydrogenase and myokinase with a reactive AMP analog.

Rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD) and myokinase (MK) were rapidly inactivated by a reactive AMP analog, N6-(p-bromoacetaminobenzyl)-AMP, under mild conditions. Complete inactivation was observed when 4 and 0.3 mol of the reagent with respect to enzyme were reacted with GPD and MK, respectively. The inactivation of both enzymes were favored at higher pH and the enzymes were protected by addition of adenine nucleotide substrate. Modified GPD or MK had no affinity for AMP-Sepharose, in contrast to the native enzymes. From these results, the inactivation of GPD and MK by the reactive AMP analog can be regarded as an affinity labeling. The posibility that the present AMP analog may be used as a general affinity labeling reagent for various adenine nucleotide-related enzymes is discussed based on the results obtained.     

Binding of adenine nucleotides to the F1-inhibitor protein complex of bovine heart submitochondrial particles.

The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of rabbit muscle phosphorylase b by a water-soluble carbodiimide.

Glycogen phosphorylase b from rabbit muscle was rapidly inactivated by incubation with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC) at pH 5.1. The inactivation was pH-dependent and was not restored by treatment with hydroxylamine. The addition of glycine ethyl ester or N-(2,4-dinitrophenyl)-ethylenediamine (DNP-EDA) markedly increased the rate of inactivation. Of the various amino analogs of glucose tested, only glucosyl amine accelerated the inactivation, although they are all bound to the glucose 1-phosphate site of the enzyme. In the absence of amines, incorporation of about 3 mol of [metho-14C]CMC per protein monomer was observed on complete inactivation. In the presence of DNP-EDA, however, only 2 mol of [metho-14C]CMC and 1 mol of DNP-EDA were incorporated before the activity was completely lost. The treatment of phosphorylase b with CMC did not change the Km values of the enzyme for glucose 1-phosphate and AMP, in spite of the 56% inactivation. It is suggested that, in the phosphorylase-catalyzed reaction, an essential carboxyl group of the enzyme plays a role in the protonation of the glucosidic oxygen of glucose 1-phosphate.     

Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate

2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme

Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme (Mr = 84,000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of epsilon-N-pyridoxyllysine. Quantitative determinations established that 2 mol of pyridoxal 5'-phosphate are bound per mol of dimeric enzyme. After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Lys*-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly -Tyr-Thr-Ala-Ala-Arg (where * = the lysine residue in azomethine linkage with pyridoxal 5'-phosphate). This peptide corresponds to residues 235-257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver.     

The reaction of bovine glutamate dehydrogenase with periodate-oxidised ADP

1. The reactive analogue oADP produced by periodate oxidation of ADP has been studied as a potential affinity label for the enzyme bovine glutamate dehydrogenase, using circular dichroism (CD) difference spectroscopy to monitor specific binding. 2. The analogue binds stoichiometrically, rapidly and reversibly to the adenine nucleotide binding site with Kd approximately equal to 12 microM (20 degrees C, pH 7) with characteristic intensification of the adenine nucleotide CD at 260 nm. 3. This complex is unstable and decays with a half-life of about 1.5 h; the analogue then becomes attached as a Schiff base to a number of subsidiary sites, including the enzyme active site, with partial inactivation of the enzyme. 4. Depending upon initial concentration of oADP, the enzyme activity is progressively lost during the slow reaction; following borohydride reduction, up to four molecules of analogue are bound/subunit. 5. Protection against loss of enzyme activity is afforded by the coenzyme NAD+ plus glutarate or L-hydroxyglutarate (an effective inhibitor), or by glutarate alone, but not by NAD+ alone. 6. Spectroscopic and protection studies indicate that after the decay of the specific CD signal, the enzyme retains the capacity to bind ADP, but that this is progressively lost in parallel with decay of enzymic activity. 7. The results are consistent with proximity or functional interaction between the adenine nucleotide site and the coenzyme binding portion of the active site. 8. Thus oADP does not act as a true affinity label for the adenine nucleotide binding site, but the reaction subsequent to binding at that site shows some specificity directed towards the active site.     

Affinity labeling of the catalytic and AMP allosteric sites of 3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase by 5'-p-fluorosulfonylbenzoyladenosine

The nucleotide analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) reacts irreversibly with rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase kinase, causing a rapid loss of the AMP activation capacity and a slower inactivation of the catalytic activity. The rate constant for loss of AMP activation is about 10 times higher (kappa 1 = 0.112 min-1) than the rate constant of inactivation (kappa 2 = 0.0106 min-1). There is a good correspondence between the time-dependent inactivation of reductase kinase and the time-dependent incorporation of 5'-p-sulfonylbenzoyl[14C]adenosine ([14C]SBA). An average of 1.65 mol of reagent/mol of enzyme subunit is bound when reductase kinase is completely inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 1 mol of SBA/mol of subunit causes complete loss of AMP activation, whereas reaction of another mole of SBA/mol of subunit would lead to total inactivation. Protection against inactivation by the reagent is provided by the addition of Mg2+, AMP, Mg-ATP, or Mg-AMP to the incubation mixtures. In contrast, addition of ATP, 2'-AMP, or 3'-AMP has no effect on the rate constants. Mg-ATP protects preferentially the catalytic site against inactivation, whereas Mg-AMP at low concentration protects preferentially the allosteric site. Mg-ADP affords less protection than Mg-AMP to the allosteric site when both nucleotides are present at a concentration of 50 microM with 7.5 mM Mg2+. Experiments done with [14C]FSBA in the presence of some protectants have shown that a close correlation exists between the pattern of protection observed and the binding of [14C]SBA. The postulate is that there exists a catalytic site and an allosteric site in the reductase kinase subunit and that Mg-AMP is the main allosteric activator of the enzyme.     

Modification of NAD-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivatives of NAD, NADH, NADP, and NADPH

The 2',3'-dialdehyde nicotinamide ribose derivatives of NAD (oNAD) and NADH (oNADH) have been prepared enzymatically from the corresponding 2',3'-dialdehyde analogs of NADP and NADPH. Pig heart NAD-dependent isocitrate dehydrogenase requires NAD as coenzyme but binds NADPH, as well as NADH, ADP, and ATP, at regulatory sites. Incubation of 1-3 mM oNAD or oNADH with this isocitrate dehydrogenase causes a time-dependent decrease in activity to a limiting value 40% that of the initial enzyme, suggesting that reaction does not occur at the catalytic coenzyme site. Upon varying the concentration of oNAD or oNADH from 0.2 to 3 mM, the inactivation rate constants increase in a nonlinear manner, consistent with reversible binding of oNAD and oNADH to the enzyme prior to covalent reaction. Inactivation is accompanied by incorporation of radioactive reagent with extrapolation to 0.54 mol [14C]oNAD or 0.45 mol [14C]oNADH/mol average enzyme subunit (or about 2 mol reagent/mol enzyme tetramer) when the enzyme is maximally inactivated; this value corresponds to the number of reversible binding sites for each of the natural ligands of isocitrate dehydrogenase. The protection against oNAD or oNADH inactivation by NADH, NADPH, and ADP (but not by isocitrate, NAD, or NADP) indicates that reaction occurs in the region of a nucleotide regulatory site. In contrast to the effects of oNAD and oNADH, oNADP and oNADPH cause total inactivation of the NAD-dependent isocitrate dehydrogenase, concomitant with incorporation, respectively, of about 3.5 mol [14C]oNADP or 1.3 mol [14C]oNADPH/mol average subunit. Reaction rates exhibit a linear dependence on [oNADP] or [oNADPH] and protection by natural ligands against inactivation is not striking. These results imply that oNADP and oNADPH are acting in this case as general chemical modifiers and indicate the importance of the free adenosine 2'-OH of oNAD and oNADH for specific labeling of the NAD-dependent isocitrate dehydrogenase. The new availability of 2',3'-dialdehyde nicotinamide ribose derivatives of NAD, NADH, NADP, and NADPH may allow selection of the appropriate reactive coenzyme analog for affinity labeling of a variety of dehydrogenases.     

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.     

3(17)beta-Hydroxysteroid Dehydrogenase of Pseudomonas testosteroni. Ligand binding properties.

The binding of NAD and NADH to electrophoretically pure 3(17)beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni was determined by Fluorescence spectroscopy and gel filtration. Four moles of cofactor are bound/mol of tetrameric enzyme; the binding sites are equivalent and independent. The dissociation constants for NAD and NADH are 16 and 0.25 micronM, respectively. As measured by gel filtration in the absence of cofactor, 0.4 mol of estradiol-17 beta is bound/mol of tetrameric enzyme. Data obtained from isotope exchange at equilibrium indicate that the binding of the cofactor to the enzyme is favored over the binding of steroid, although each may bind in the absence of the other. The rates of cofactor dissociation from the ternary complexes are slower than the rates of steroid dissociation; cofactor dissociation is probably the rate-limiting step. Cofactor analogs modified in the pyridine moiety are cosubstrates, whereas modified adenine derivatives are not. The enzyme also utilized as substrate a number of potential steroid affinity labels; no enzyme inactivation by these compounds was observed.