The ATPase activity of the alpha 3 beta 3 complex of the F1-ATPase of the thermophilic bacterium PS3 is inactivated on modification of tyrosine 307 in a single beta subunit by 7-chloro-4-nitrobenzofurazan

The catalytically active alpha 3 beta 3 complex, assembled as described (Miwa, K., and Yoshida, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6484-6487) from the isolated alpha and beta subunits of the F1-ATPase of the thermophilic bacterium PS3 (TF1), is inactivated by 7-chloro-4-nitrobenzofurazan (Nbf-Cl) with characteristics very similar to those observed when TF1, which has the subunit composition, alpha 3 beta 3 gamma delta epsilon, is inactivated by the reagent under the same conditions. Both native TF1 and the alpha 3 beta 3 complex are inactivated by 200 microM Nbf-Cl with a pseudo-first order rate constant of 3.7 x 10(-2) min-1 in the presence of 0.2 M Na2SO4 at pH 7.6 and 23 degrees C. The rate of increase in absorbance at 385 nm of reaction mixtures containing 200 microM [14C]Nbf-Cl and TF1, the wild-type alpha 3 beta 3 complex, or the mutant alpha 3(beta Y307----F)3 complex, each at 18 microM was also examined. Since the alpha 3(beta y307----F)3 complex is resistant to inactivation by Nbf-Cl, difference spectrophotometry revealed that inactivation of native TF1 and the wild-type alpha 3 beta 3 complex could be correlated with formation of about 1 mol of Nbf-O-Tyr/mol of enzyme or complex. Fractionation of peptic digests of the labeled enzyme and complexes by reversed-phase high performance liquid chromatography resolved a major radioactive peptide that was common to labeled TF1 and the labeled alpha 3 beta 3 complex but was absent in the digest of the labeled alpha 3(beta Y307----F)3 complex. This labeled peptide was shown to contain Tyr-beta 307 derivatized with [14C]Nbf-Cl by automatic amino acid sequence analyses. From these results, it is concluded that one-third of the sites' reactivity of Nbf-Cl with Tyr-beta 307 in TF1 or its equivalent in other F1-ATPases is not influenced by the presence of the gamma, delta, or epsilon subunits. It has also been shown that Tyr-307 is not modified to an appreciable extent when the isolated beta subunit is treated with [14C]Nbf-Cl under conditions in which this residue is nearly completely labeled in a single beta subunit when TF1 or the alpha 3 beta 3 complex is inactivated by the reagent.     

Selective activation of cyclic AMP dependent protein kinase by calcitonin in a calcitonin secreting lung cancer cell line

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.3 and then gel-filtered, 1.25 mols of [14C]Nbf-O-Tyr and less than 0.1 mol of Nbf-N-Lys were formed per mol of enzyme. After adjusting the pH of the gel-filtered, modified enzyme to 9.0 and incubating it for 14 hrs. at 23 degrees C to promote O----N migration, 0.68 mol of Nbf-N-Lys were formed per mol of enzyme while about 16% of the original activity reappeared. Isolation of the subunits after the O----N migration showed that 90% of the incorporated 14C was present in the beta subunit, which contained 0.21 mols of [14C]Nbf-N-Lys per mol. A tryptic peptide which contained the majority of the 14C incorporated into the beta subunit was isolated and subjected to automatic amino acid sequence analysis contained 38 residues. The amino acid sequence immediately around the lysine residue labeled with [14C]Nbf-, K*, was found to be: ...I-G-L-F-G-G-A-G-V-G-K*-T-V-L-I-G... .     

Separate beta subunits are derivatized with 14C and 3H when the bovine heart mitochondrial F1-ATPase is doubly labeled with 7-chloro-4-nitro[14C]benzofurazan and 5'-p-fluorosulfonylbenzoyl[3H]inosine.

Tyrosine residues 311 and 345 of the beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) are present on the same peptide when the enzyme is fragmented with cyanogen bromide. Maximal inactivation of MF1 with 7-chloro-4-nitro[14C]benzofurazan [( 14C]Nbf-Cl) derivatizes tyrosine-311 in a single beta subunit. Cyanogen bromide digests of MF1 containing the [14C]Nbf-O-derivative of tyrosine-beta 311 were submitted to reversed-phase HPLC, with and without prior reduction of the nitro group on the incorporated reagent with dithionite. The retention time of the radioactive cyanogen bromide peptide was shifted substantially by reduction. When a cyanogen bromide digest of MF1 inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine [( 3H]FSBI), which proceeds with derivatization of tyrosine-345 in a single beta subunit, was submitted to HPLC under the same conditions, the fragment labeled with 3H eluted with the same retention time as the [14C]Nbf-O-derivative before reduction. Doubly labeled enzyme was prepared by first derivatizing Tyr-beta 311 with [14C]Nbf-Cl and then derivatizing tyrosine-beta 345 with [3H]FSBI with and without reducing the [14C]Nbf-O-derivative of tyrosine-beta 311 with dithionite before modification with [3H]FSBI. The doubly labeled enzyme preparations were digested with cyanogen bromide and submitted to HPLC. The 14C and 3H in the cyanogen bromide digest prepared from doubly labeled enzyme not submitted to reduction eluted together. In contrast, the 14C and 3H in the digest prepared from doubly labeled enzyme which had been reduced eluted separately. From these results it is concluded that different beta subunits are derivatized when MF1 is doubly labeled with [14C]Nbf-Cl and [3H]FSBI.     

On the subunit stoichiometry of the F1-ATPase and the sites in it that react specifically with p-fluorosulfonylbenzoyl-5'-adenosine.

During the inactivation of the nucleotide-free F1-ATPase at pH 7.0, by p-fluorosulfonyl[14C]benzoyl-5'-adenosine ([14C]FSBA) in the presence of 20% glycerol, about 4.5 g atoms of 14C are incorporated/350,000 g of enzyme. Isolation of the subunits has shown: (a) over 90% of the incorporated label is associated with the alpha and beta subunits; (b) the amount of label incorporated into the alpha subunit is about 0.5 g atoms/mol which is nonspecifically associated with a number of tyrosine and lysine residues; (c) the amount of radioactivity incorporated into the beta subunit is about 0.9 g atoms/mol which correlates with the degree of inactivation of the enzyme and resides on a single tyrosine residue; (d) up to 2.2 mol of alpha subunit have been isolated from each mole of inactivated enzyme; and (e) about 2 mol of beta subunit have been isolated from each mole of inactivated enzyme. These results account for the incorporation of 4.5 g atoms of 14C which are incorporated/mol of ATPase during inactivation if there are three copies each of the alpha and beta subunit present in the enzyme. It has also been shown that 4-chloro-7-nitrobenzofurazan (NBD-Cl) and FSBA react with different tyrosine residues when they inactivate the ATPase. In addition, it has been shown that the ATPase inactivated with FSBA retains the capacity to bind up to 2.2 mol of [14C]ADP/350,000 g of enzyme.     

Photoaffinity labeling of the tight ADP binding site of the chloroplast coupling factor one (CF1): the effect on the CF1-ATPase activity

Chloroplast thylakoid membranes contain tightly bound ADP which is intimately involved in the mechanism of photophosphorylation. The photoaffinity analog 2-azido-ADP binds tightly to spinach thylakoid membrane-bound coupling factor one (CF1) and, in a manner similar to ADP, inhibits the light-triggered ATPase activity (Czarnecki, J.J., Abbott, M.S. and Selman, B.R. (1983) Eur. J. Biochem. 136, 19-24). Ultraviolet irradiation of thylakoid membranes containing noncovalently, tightly bound 2-azido[beta-32P]ADP results in the inactivation of both the methanol-stimulated MgATPase activity of the membrane-bound CF1 and the octylglucoside-dependent MgATPase activity of the solubilized enzyme. There is a linear correlation between the loss of enzyme activity and the covalent incorporation of the photoaffinity analog. Full inactivation of catalytic activity is estimated to occur upon incorporation of 1.07 mol analog and 0.65 mol analog per mol enzyme for the methanol- and octylglucoside-stimulated activities, respectively. Since 2-azido-ADP modifies only the beta subunit of the CF1 and since there are probably three beta subunits per CF1, these results indicate strong cooperativity among beta subunits and between the site of tightly bound nucleotides and the catalytic sites.     

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.     

Identification of an essential glutamic acid residue in the beta subunit of the adenosine triphosphatase from the thermophilic bacterium PS3

The TF1-ATPase from the thermophilic bacterium, PS3, is inactivated by dicyclohexylcarbodiimide (DCCD). This inactivation is stimulated by ADP and other specific nucleotides and is inhibited by Mg2+. When the inactivation is carried out with [14C]DCCD, about 2 g atoms of 14C are bound/mol of TF1 when the enzyme is nearly completely inactivated. The isolated subunits from TF1 inactivated with [14C]DCCD contain the following amounts of 14C/mol: alpha, 0.12 g atom; beta, 0.47 g atom; gamma, approximately 0.04 g atom; delta, none; and epsilon, 0.05 g atom. Fractionation of tryptic digests have shown that the 14C bound to the alpha subunit is nonspecifically associated with several peptides, and that the 14C bound to the beta subunit is associated with a single tryptic peptide with the amino acid sequence Ala-Gly-Val-Gly-Glu-Arg, where Glu represents the N-gamma-glutamyl derivative of dicyclohexyl[14C]urea.     

Identification of an essential arginine residue in the beta subunit of the chloroplast ATPase

The ATPase activity of soluble chloroplast coupling factor (CF1) was irreversibly inactivated by phenylglyoxal, an arginine reagent. Under the conditions of inactivation, 2.48 mol of [14C]phenylglyoxal were incorporated per 400,000 g of enzyme when the ATPase was inactivated 50% by the reagent. Isolation of the component polypeptide subunits of the [14C]phenylglyoxal-modified enzyme revealed that the distribution of moles of labeled reagent/mol of subunit was the following: alpha, 0.37; beta, 0.40; gamma, 0.08; delta, none; epsilon, 0.03. CNBr treatment of the isolated alpha and beta subunits and fractionation of the peptides by gel electrophoresis revealed that the radioactivity bound to the alpha subunit was nonspecifically associated with several peptides, while a single peptide derived from the beta subunit contained the majority of the radioactivity associated with this subunit. After treating the isolated beta subunit with trypsin and Staphylococcus aureus protease, a major radioactive peptide was isolated with a sequence Arg-Ile-Thr-Ser-Ile-Lys. This sequence, when compared with the primary structure of the CF1 beta subunit as translated from the gene (Zurawski, G., Bottomley, W., and Whitfeld, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6260-6264) indicated that the arginine marked with the asterisk, the predominant residue modified by phenylglyoxal when the ATPase activity of CF1 is inactivated by the reagent, is Arg 312.     

The interaction of an epoxide with yeast alcohol dehydrogenase: evidence for binding and the modification of two active site cysteines by styrene oxide.

Yeast alcohol dehydrogenase is inactivated and alkylated by styrene oxide in a single exponential kinetic process. The concentration dependence of half-times for inactivation indicates the formation of an enzyme inhibitor complex, KI = 2.5 times 10(-2) M at pH 8.0. Reduced nicotinamide adenine dinucleotide (NADH), at a concentration of 3 times 10(-4) M where Kd congruent to 1 times 10(-5) M, has a small effect on kinetic parameters for inactivation. Although benzyl alcohol and acetamide-NADH increase the KI for styrene oxide in a manner consistent with their dissociation constants, substrate also increases the rate of inactivation at high styrene oxide concentrations. The reciprocal of half-times for inactivation, extrapolated to infinite styrene oxide concentration, increases with pH between 7.6 and 9.0, pK congruent to 8.5. The stoichiometry of alkylation by [3H]styrene oxide is 2.2 mol of reagent incorporated/mol of subunit, and is accompanied by the loss of 1.9 mol of sulfhydryl/mol of subunit; prior alkylation with iodoacetamide reduces the stoichiometry to 0.88:1, and increases the rate of labeling. Tryptic digests of enzyme modified with [14C]iodoacetamide or [3H]styrene oxide produce two major peptides which cochromatograph, indicating that styrene oxide and iodoacetamide modify the same cysteine residues. Previous investigators have reported that iodoacetate, iodoacetamide, and butyl isocyanate alkylate either of two reactive cysteines of yeast alcohol dehydrogenase; both cysteines cannot be modified simultaneously [Belke et al. (1974), Biochemistry 13, 3418]. The inactivation of enzyme by p-chloromercuribenzoate (PCMB) is reported here to be accompanied by the incorporation of 2.3 mol of PCMB/mol of enzyme subunits, in analogy with styrene oxide; the planarity of the alkylating agent appears to be an important factor in determining the stoichiometry of labeling.     

The use of dithionite reduction to identify the essential tyrosine residue in the F1-ATPase from the thermophilic bacterium, PS3, that reacts with 7-chloro-4-nitrobenzofurazan

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by greater than 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.4, 1.4 mol of [14C]Nbf were incorporated per mol of enzyme. After pepsin digestion of the labeled enzyme at pH 3.0, a single, major peak of radioactivity was resolved by reversed-phase high-performance liquid chromatography under acidic conditions were peptidyl Nbf-O-tyrosine is stable. This radioactive peak, designated RP-1, eluted with a retention time of 95 min. When the material in RP-1 was subjected to reversed-phase high-performance liquid chromatography under the same conditions after treatment with sodium dithionite, a single, major peak of radioactivity, designated RP-2, was resolved with a retention time of 52 min. Automatic Edman degradation of this material revealed that it has the amino acid sequence I-Y*-V-P-A-D-(D), where Y* presumably represents peptidyl [14C]Nbf-O-tyrosine. These results provide the basis for a facile method to purify peptides containing [14C]Nbf-O-tyrosine in which the labeled residues can be identified by amino acid sequence analysis using the Edman degradation.     

Identification of the essential tyrosine residue in the beta subunit of bovine heart mitochondrial F1-ATPase that is modified by 7-chloro-4-nitro[14C]benzofurazan

Inactivation of the bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.5, led to the incorporation of 1.42 g atoms of 14C/mol. Treatment of the inactivated enzyme with dithiothreitol removed 0.99 g atom of 14C/mol of enzyme which was accompanied by reactivation of the ATPase. Therefore, of the 1.42 mol of 7-chloro-4-nitro-[14C]benzofurazan incorporated per mol of bovine heart mitochondrial F1-ATPase, 0.43 mol was present on lysine residues and 0.99 mol was present on tyrosine residues. When the inactivated enzyme was treated with 10 mM sodium dithionite at pH 6.0, 10% of the activity was recovered which was accompanied by a 10% loss in covalently bound 14C. Following dithionite treatment, that part of the 14C which remained covalently bound could not be removed by subsequent treatment of the labeled enzyme with dithiothreitol. It is presumed that dithionite reduces the 4-nitro group of the covalently bound reagent, converting it to 4-amino[14C]benzofurazan derivatives at lysine and tyrosine residues. The moles of 4-amino[14C]benzofurazan incorporated per mol of the isolated subunits were: alpha, 0.18; beta, 0.30; gamma, 0.03; and delta plus epsilon, less than 0.01. Gel filtration of a cyanogen bromide digest of the labeled beta subunit on Sephadex G-75 resolved a major 14C peak which contained 83% of the 14C recovered. The major, radioactive tryptic fragment derived from this peak was purified by gel filtration on Sephadex G-75 followed by reversed phase high performance liquid chromatography. Automatic Edman degradation of this peptide showed that the 14C was released at the position occupied by beta-Tyr-311.     

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)     

Sequence of the radioactive tryptic peptide obtained after inactivating the F1-ATPase of the thermophilic bacterium PS3 with 5'-p-fluorosulfonylbenzoyl[3H]adenosine at 65 degrees C

Following a lag of about 30 min, the F1-ATPase from the thermophilic bacterium, PS3 (TF1), was inactivated slowly by 0.8 mM 5'-p-fluorosulfonylbenzoyladenosine (FSBA) at 23 degrees C and pH 7.0. When the enzyme was treated with 0.2 mM FSBA at pH 7.0 and 23 degrees C for 15 min and gel-filtered, no enzyme activity was lost. However, the lag in inactivation was abolished when the enzyme was subsequently incubated with 2.0 mM FSBA at 23 degrees C in the pH range from 6.8 to 10.0. The pH-inactivation profile obtained under these conditions revealed a pK alpha of about 9.3 which was associated with the inactivation. When pretreated TF1 was inactivated at 23 degrees C with [3H]FSBA by about 90%, greater than 20 mol of [3H]SBA was incorporated per mole of enzyme. TF1 was inactivated rapidly by 0.8 mM FSBA at pH 6.4 and 65 degrees C, and no lag was observed. Following inactivation of TF1 with 0.8 mM [3H]FSBA at 65 degrees C and pH 6.4, about 10 mol of [3H]SBA was incorporated per mole of enzyme. When a tryptic digest of the labeled enzyme was fractionated by reversed-phase high-performance liquid chromatography, a single major radioactive peptide was isolated. When subjected to automatic Edman degradation, this peptide was shown to have the amino acid sequence: A-L-A-P-E-I-V-G-E-E-H-X-Q-V-A-R, where X indicates that a phenylthiohydantoin derivative was not detected in cycle 12. However, from the DNA sequence of the gene encoding the subunit of TF1 (Y. Kagawa, M. Ishizuka, T. Saishu, and S. Nakao (1985) Abstracts International Symposium on Energy Transducing ATPases, Kobe, Japan, p. 84), this position has been shown to be occupied by tyrosine. This tyrosine is homologous with beta-Tyr-368 of the bovine mitochondrial F1-ATPase (MF1) the modification of which is responsible for the inactivation MF1 by FSBA.     

Affinity labeling of the pyridoxal phosphate binding site of the beta2 subunit of Escherichia coli tryptophan synthase.

We have synthesized bromoacetylpyridoxamine phosphate and bromoacetylpyridoxamine and have shown that they meet three criteria for affinity labels of the beta2 subunit of tryptophan synthase: (i) the kinetic data of inactivation indicate that a binary complex is formed prior to covalent attachment; (ii) inactivation is largely prevented by the presence of pyridoxal phosphate; and (iii) inactivation is stoichiometric with incorporation of 0.7 to 0.8 mol of chromophore/mol of beta monomer. Our conclusion that inactivation of the apo beta2 subunit by bromoacetylpyridoxamine phosphate is due to the modification of cysteine is based on the disappearance of 1 mol of -SH/beta monomer and on the finding that [14C]carboxymethyl derivative in the acid hydrolysate of the protein modified by bromo[14C]acetylpyridixamine phosphate. A 39-residue tryptic peptide containing this essential cysteine has been isolated and purified from the bromo[14C]acetylpyridoxamine phosphate-labeled beta2 subunit.     

Chemical modification of thiol groups of mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe. Involvement of alpha- and gamma-subunits in the enzyme activity

Mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe has been prepared under a stable form and in relatively high amounts by an improved purification procedure. Specific chemical modification of the enzyme by the thiol reagent N-ethylmaleimide (NEM) at pH 6.8 leads to complete inactivation characterized by complex kinetics and pH dependence, indicating that several thiols are related to the enzyme activity. A complete protection against NEM effect is afforded by low concentrations of nucleotides in the presence of Mg2+, with ADP and ATP being more efficient than GTP. A total binding of 5 mol of [14C]NEM/mol of F1-ATPase is obtained when the enzyme is 85% inactivated: 3 mol of the label are located on the alpha-subunits and 2 on the gamma-subunit. Two out of the 3 mol on the alpha-subunits bind very rapidly before any inactivation occurs, indicating that the two thiols modified are unrelated to the inactivation process. Complete protection by ATP against inactivation by NEM prevents the modification of three essential thiols out of the group of five thiols labeled in the absence of ATP: one is located on a alpha-subunit and two on the gamma-subunit. These two essential thiols of the gamma-subunit can be differentiated by modification with 6,6'-dithiodinicotinic acid (CPDS), another specific thiol reagent. A maximal binding of 4 mol of [14C]CPDS/mol of enzyme is obtained, concomitant to a 25% inhibition. Sequential modification of the enzyme by CPDS and [14C]NEM leads to the same final deep inactivation as that obtained with [14C]NEM alone. One out of the two thiols of the gamma-subunit is no longer accessible to [14C]NEM after CPDS treatment. When incubated at pH 6.8 with [3H]ATP in the presence of Mg2+, F1-ATPase is able to bind 3, largely exchangeable, mol of nucleotide/mol of enzyme. Modification of the three essential thiols by NEM dramatically decreases the binding of 3H-nucleotide down to about 1 mol/mol of enzyme. Partial modification modifies the cooperative properties, the enzyme being no longer sensitive to anion activation.     

Purification and kinetic and structural properties of spinach leaf NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase

NADP-dependent nonphosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation, molecular sieving on Sephadex G-200, DEAE-cellulose, and 2',5'-ADP-Sepharose affinity chromatography. The purified enzyme exhibited a specific activity of 15 mumol (mg protein)-1 min-1 and was characterized as a homotetramer with a native molecular weight of 195,000. Preincubation of the purified enzyme with NADP+ resulted in an almost twofold increase in enzymatic activity. The rate of activation was slower than the rate of catalysis, indicating that the enzyme has hysteretic properties. This behavior results in a lag phase during activity measurement of the enzyme preincubated without NADP+. Substrate interaction and product inhibition studies suggest a rapid equilibrium random BiBi mechanism for the reaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivated the purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kinetics with a rate constant of 0.17 min-1. D-Glyceraldehyde 3-phosphate effectively protected the enzyme against inactivation by thiol reagents, suggesting that modification occurred at or near the substrate-binding site. Complete inactivation of the dehydrogenase was correlated with incorporation of 8 mol [1-14C]iodoacetamide/mol enzyme. Total protection afforded by D-glyceraldehyde 3-phosphate against enzyme inactivation by iodoacetamide was correlated with a protection of 4 mol reactive residues/mol enzyme. On the basis of these results it is suggested that one sulfhydryl group per enzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reaction is proposed.     

5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli--the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411

In order to identify the essential reactive amino acid residues of 5-enolpyruvylshikimate-3-phosphate synthase, the reaction of the enzyme with its substrate analogue bromopyruvate was investigated. Incubation of the enzyme with bromopyruvate resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact of 28 microM and a maximum rate constant of 0.31 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvylshikimate 3-phosphate or by the combination of shikimate 3-phosphate plus glyphosate (N-phosphonomethylglycine), an inhibitor of the enzyme. Addition of sodium [3H]borohydride to the reaction mixture had no effect on the rate of inactivation but resulted in the incorporation of 3H label to the modified enzyme. Upon 90% inactivation, approximately 1 mol of bromo[14C]pyruvate was incorporated per mole of enzyme modified in the absence or presence of sodium borohydride. When the enzyme was incubated with bromopyruvate in the presence of sodium [3H]borohydride, approximately 1 mol of 3H label was found to be associated per mole of the modified enzyme. Tryptic digestion of these labeled proteins followed by reverse phase chromatographic separation resulted in the isolation of three radioactive peptides. Analyses of these three peptides indicated that bromopyruvate inactivated the enzyme by modifying Cys-408 and Lys-411, which are conserved in all enzyme sequences studied to date.     

Identification of the lysine residue to which the 4-nitrobenzofurazan group migrates after the bovine mitochondrial F1-ATPase is inactivated with 7-chloro-4-nitro[14C]benzofurazan

When bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, was inactivated by greater than 90% with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.4, 1.15 mol of 4-nitrobenzofurazan [14C]Nbf were incorporated per mol of enzyme. Reactivation of a sample of the modified enzyme with dithiothreitol removed 0.82 mol of [14C]Nbf/mol of the F1-ATPase indicating that, of the 1.15 mol of [14C]Nbf incorporated, 0.82 mol were present on tyrosine residues and 0.33 mol on lysine residues. Incubation of the modified enzyme at pH 9.0 for 18 h at 23 degrees C led to an increase of 0.64 mol of [14C]Nbf-N'-Lys/mol of the F1-ATPase which occurred as a consequence of an O----N migration. About 15% enzyme reactivation occurred simultaneously with the migration indicating that the fraction of the [14C]Nbf group originally present on tyrosine which did not migrate was lost by hydrolysis. Examination of a tryptic digest of the labeled enzyme after the O----N migration by reversed-phase high-pressure liquid chromatography revealed a single major radioactive peptide. The labeled tryptic fragment was purified and subjected to automatic Edman degradation. This analysis revealed that Lys-beta-162 was specifically labeled during the O----N migration of the [14C]Nbf group.     

Characterization of exchangeable and nonexchangeable bound adenine nucleotides in F1-ATPase from Escherichia coli

The total amount of bound exchangeable and nonexchangeable adenine nucleotides in Escherichia coli F1-ATPase (BF1) was determined; three exchangeable nucleotides were assessed by equilibrium dialysis in a [14C]ADP-supplemented medium. When BF1 was purified in a medium supplemented with ATP, a stoichiometry of nearly 6 mol of bound nucleotides/mol of enzyme was found; three of the bound nucleotides were ATP and the others ADP. When BF1 was filtered on Sephadex G-50 in a glycerol medium (Garrett, N.E., and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647), bound ADP was rapidly released, in contrast to bound ATP which remained firmly attached to the enzyme. Upon incubation of BF1 with [14C]ADP, the bound ADP rather than the bound ATP was exchanged. Of the three [14C]ADPs which have bound to BF1 by exchange after equilibrium dialysis, one was readily lost by gel filtration on Sephadex G-50; the loss of bound [14C]ADP was markedly reduced by incubation of BF1 with aurovertin, a specific ligand of the beta subunit which is known to increase the affinity of the beta subunit for nucleotides (Issartel, J.-P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). Upon photoirradiation of BF1 with [alpha-32P]2-azido-ADP, only the beta subunit was labeled; concomitantly, bound ADP was released, but the content in bound ATP remained stable. These results suggest that specific sites located on the three beta subunits bind nucleotides in a reversible manner. Consequently, the tightly bound ATP of native BF1 would be located on the alpha subunits.     

Inactivation of beef heart mitochondrial F1-ATPase by the 2',3'-dialdehyde derivatives of adenine nucleotides

Beef heart mitochondrial F1-ATPase was inactivated by the 2',3'-dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol F1. After correction for non-specific labeling, the number of specifically bound [14C]oADP was 2-3 mol per mol F1. By SDS-polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the alpha and beta subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.