Identification of amino acid residues involved in substrate recognition by the catalytic subunit of bovine cyclic AMP dependent protein kinase: peptide-based affinity labels

Two peptide-based affinity inactivators Ac-Leu-(BrAc)Orn-Arg-Ala-Ser-Leu-Gly (4) and Ac-Leu-Arg-(BrAc)Orn-Ala-Ser-Leu-Gly (5) were prepared as probes for the study of the nature of the active-site residues in the catalytic subunit of cyclic AMP dependent protein kinase. Under conditions of inhibitor in excess, both peptides inactivated the catalytic subunit by an apparent biphasic process. A fast phase, which inactivated the protein by approximately 40%, was followed by a slow phase that accounted for the loss of the remaining enzyme activity. Protection experiments with the kinase substrates showed that the slow phase of inactivation was active site directed, while the fast phase was not. Studies with radioactively labeled peptides 4 and 5 indicated incorporation of two peptide residues per molecule of the catalytic subunit upon complete inactivation. This observation is consistent with the occurrence of one alkylation event in each phase of the inactivation. The protein was proteolyzed subsequent to its modification with radioactive peptides. High-performance liquid chromatography afforded two radioactive peptide fragments in each case, which were sequenced by Edman degradation. Peptide 4 alkylated Thr-197 and Glu-346, while peptide 5 modified Cys-199 and also Glu-346. Data are presented to support the conclusion that Thr-197 and Cys-199 are located at or near the active site.     

Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Identification of a specific interchain disulfide bond

The catalytic (C) subunit and the type II regulatory (RII) subunit of cAMP-dependent protein kinase can be cross-linked by interchain disulfide bonding. This disulfide bond can be catalyzed by cupric phenanthroline and also can be generated by a disulfide interchange using either RII-subunit or C-subunit that has been modified with either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-4(azidophenylthio)phthalimide (APTP). When the 2 cysteine residues of the C-subunit are reacted with DTNB prior to incubation with the RII-subunit, interchain disulfide bonding occurs. Similar observations are seen with C-subunit that had been modified with APTP. Interchain disulfide bonds also form when the RII-subunit is modified with DTNB prior to incubation with the C-subunit. The presence of cAMP facilitates this cross-linking while autophosphorylation of the RII-subunit retards the rate at which the interchain disulfide bond forms. Interchain disulfide bonds also form spontaneously when the RII-subunit and the C-subunit are dialyzed at pH 8.0 in the absence of reducing agents. The specific amino acid residues that participate in intersubunit disulfide bonding have been identified as Cys-97 in the RII-subunit and Cys-199 in the C-subunit. Based on the sequence homologies of the RII-subunit with other kinase substrates and on the proximity of Cys-97 to the catalytic site, a model is proposed in which the autophosphorylation site of the RII-subunit occupies the substrate-binding site in the holoenzyme. The model also proposes that this same site may be occupied by the region flanking Cys-199 in the C-subunit when the C-subunit is dissociated.     

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: fluorescent affinity labeling of the catalytic subunit from bovine skeletal muscle with o-phthalaldehyde

The catalytic subunit of adenosine cyclic 3',5'-monophosphate dependent protein kinase from bovine skeletal muscle was rapidly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics, and the second-order rate constant was 1.1 X 10(2) M-1 s-1. Absorbance and fluorescence spectroscopic data were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme). The reaction between the catalytic subunit and o-phthalaldehyde was not reversed by the addition of reagents containing free primary amino and sulfhydryl functions following inactivation. The reaction, however, could be arrested at any stage during its progress by the addition of an excess of cysteine or less efficiently by homocysteine or glutathione. The catalytic subunit was protected from inactivation by the presence of the substrates magnesium adenosine triphosphate and an acceptor serine peptide substrate. The decrease in fluorescence emission intensity of incubation mixtures containing iodoacetamide- or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified catalytic subunit and o-phthalaldehyde paralleled the loss of phosphotransferase activity. Catalytic subunit denatured with urea failed to react with o-phthalaldehyde. Inactivation of the catalytic subunit by o-phthalaldehyde is probably due to the concomitant modification of lysine-72 and cysteine-199. The proximal distance between the epsilon-amino function of the lysine and the sulfhydryl group of the cysteine residues involved in isoindole formation in the native enzyme is estimated to be approximately 3 A. The molar transition energy of the catalytic subunit-o-phthalaldehyde adduct was 121 kJ/mol and compares favorably with a value of 127 kJ/mol for the 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl)isoindole in hexane, indicating that the active site lysine and cysteine residues involved in formation of the isoindole derivative of the catalytic subunit are located in a hydrophobic environment. o-Phthalaldehyde probably acts as an active site specific reagent for the catalytic subunit.     

Signaling the signal, cyclic AMP-dependent protein kinase inhibition by insulin-formed H2O2 and reactivation by thioredoxin

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.     

Crystal structure of the Escherichia coli peptide methionine sulphoxide reductase at 1.9 A resolution

BACKGROUND: Peptide methionine sulphoxide reductases catalyze the reduction of oxidized methionine residues in proteins. They are implicated in the defense of organisms against oxidative stress and in the regulation of processes involving peptide methionine oxidation/reduction. These enzymes are found in numerous organisms, from bacteria to mammals and plants. Their primary structure shows no significant similarity to any other known protein. RESULTS: The X-ray structure of the peptide methionine sulphoxide reductase from Escherichia coli was determined at 3 A resolution by the multiple wavelength anomalous dispersion method for the selenomethionine-substituted enzyme, and it was refined to 1.9 A resolution for the native enzyme. The 23 kDa protein is folded into an alpha/beta roll and contains a large proportion of coils. Among the three cysteine residues involved in the catalytic mechanism, Cys-51 is positioned at the N terminus of an alpha helix, in a solvent-exposed area composed of highly conserved amino acids. The two others, Cys-198 and Cys-206, are located in the C-terminal coil. CONCLUSIONS: Sequence alignments show that the overall fold of the peptide methionine sulphoxide reductase from E. coli is likely to be conserved in many species. The characteristics observed in the Cys-51 environment are in agreement with the expected accessibility of the active site of an enzyme that reduces methionine sulphoxides in various proteins. Cys-51 could be activated by the influence of an alpha helix dipole. The involvement of the two other cysteine residues in the catalytic mechanism requires a movement of the C-terminal coil. Several conserved amino acids and water molecules are discussed as potential participants in the reaction.     

Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Heterobifunctional cross-linking reagents lead to photodependent and photoindependent cross-linking

Heterobifunctional cross-linking reagents have been introduced into the catalytic subunit of cAMP-dependent protein kinase as potential probes for identifying specific points of contact between the catalytic (C)-subunit and the type II regulatory (RII) subunit in the holoenzyme complex. Since at least one of the 2 cysteine residues in the C-subunit is known to be in close proximity to the interaction site between the C-subunit and the RII-subunit, these cysteines were chosen initially as targets for covalent modification by two heterobifunctional cross-linking reagents, p-azidophenacyl bromide and N-4-(azidophenylthio)phthalimide. Treatment of the C-subunit with each reagent led to the stoichiometric modification of Cys-199 and Cys-343. In each case, the modified C-subunit was still capable of forming a stable complex with the RII-subunit. Both modified C-subunits also could be covalently cross-linked to the RII-subunit; however, the mechanisms for cross-linking differed. Catalytic subunit modified by p-azidophenacyl bromide was cross-linked to the RII-subunit in a photodependent manner by a mechanism that was maximal when holoenzyme was formed and cAMP was absent. In contrast, the C-subunit modified by N-4-(azidophenylthio)phthalimide was cross-linked to the RII-subunit by a mechanism that was independent of photolysis. In this case, cross-linking was enhanced by the presence of cAMP. This cross-linking was the result of a disulfide interchange between a modified cysteine in the C-subunit and an unmodified cysteine in the RII-subunit.     

Studies of the mechanism of action and regulation of cAMP-dependent protein kinase

Magnetic resonance and kinetic studies of the catalytic subunit of a Type II cAMP-dependent protein kinase from bovine heart have established the active complex to be an enzyme-ATP-metal bridge. The metal ion is β,γ coordinated with Δ chirality at the β-phosphorous atom. The binding of a second metal ion at the active site which bridges the enzyme to the three phosphoryl groups of ATP, partially inhibits the reaction. Binding of the metal-ATP substrate to the enzyme occurs in a diffusion-controlled reaction followed by a 40 ° change in the glycosidic torsional angle. This conformational change results from strong interaction of the nucleotide base with the enzyme. NMR studies of four ATP-utilizing enzymes show a correlation between such conformational changes and high nucleotide base specificity. Heptapeptide substrates and substrate analogs bind to the active site of the catalytic subunit at a rate significantly lower than collision frequency indicating conformational selection by the enzyme or a subsequent slow conformational change. NMR studies of the conformation of the enzyme-bound peptide substrates have ruled out α-helical and β-pleated sheet structures. The results of kinetic studies of peptide substrates in which the amino acid sequence was systematically varied were used to rule out the obligatory requirement for all possible β-turn conformations within the heptapeptide although an enzymatic preference for a β_2–5 or β_3–6 turn could not be excluded. Hence if protein kinase has an absolute requirement for a specific secondary structure, then this structure must be a coil. In the enzyme-substrate complex the distance along the reaction coordinate between the γ-P of ATP and the serine oxygen of the peptide substrate (5.3 ± 0.7 Å) allows room for a metaphosphate intermediate. This finding together with kinetic observations as well as the location of the inhibitory metal suggest a dissociative mechanism for protein kinase, although a mechanism with some associative character remains possible. Regulation of protein kinase is accomplished by competition between the regulatory subunit and peptide or protein substrates at the active site of the catalytic subunit. Thus, the regulatory subunit is found by NMR to block the binding of the peptide substrate to the active site of protein kinase but allows the binding of the nucleotide substrate and divalent cations. The dissociation constant of the regulatory subunit from the active site (10^?10m) is increased ~10-fold by phosphorylation and ~10⁴-fold by the binding of cAMP, to a value (10^?5m) which exceeds the intracellular concentration of the R₂C₂ holoenzyme complex (10^?6m). The resulting dissociation of the holoenzyme releases the catalytic subunit, permitting the active site binding of peptide or protein substrates.     

Dicyclohexylcarbodiimide cross-links two conserved residues, Asp-184 and Lys-72, at the active site of the catalytic subunit of cAMP-dependent protein kinase

In the absence of MgATP, the catalytic subunit of cAMP-dependent protein kinase is irreversibly inhibited by the hydrophobic carbodiimide dicyclohexylcarbodiimide, and this inhibition is most likely due to the formation of a cross-link between a carboxyl group and a lysine residue in the active site (Toner-Webb & Taylor, 1987). In order to identify these cross-linked residues, the catalytic subunit was modified by dicyclohexylcarbodiimide and then treated with acetic anhydride and digested with trypsin. The resulting peptides were resolved by high-performance liquid chromatography. One major absorbing tryptic peptide and one smaller peptide consistently and reproducibly showed a decrease in absorbance after the catalytic subunit had been treated with DCCD. These peptides correspond to residues 166-190 and 57-93, respectively. A unique peptide was isolated from the modified catalytic subunit, and the sequence of this peptide established that the cross-linking occurred between Asp-184 and Lys-72. The cross-linking of these two residues, which were both identified previously as essential residues, confirms the likelihood that each plays a role in the functioning of this enzyme. The fact that Asp-184 and Lys-72 appear to be invariant in all protein kinases further supports the hypothesis that these two residues, located close to one another at the active site of the enzyme, play essential roles in catalysis.     

Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit.

The active site of the cAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residues-Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241-that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.     

Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue.

The amino acid sequences of the NH2 terminus and internal peptide fragments of a Rhodococcus rhodochrous J1 nitrilase were determined to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 750-base DNA fragment thus amplified was used as the probe to clone a 5.4-kilobase PstI fragment coding for the whole nitrilase. The nitrilase gene modified in the sequence upstream from the presumed ATG start codon was expressed to approximately 50% of the total soluble protein in Escherichia coli. The predicted amino acid sequence of the nitrilase gene showed similarity to that of the bromoxynil nitrilase from Klebsiella ozaenae. The 5,5'-dithiobis(2-nitrobenzoic acid) modification of the nitrilase from R. rhodochrous J1 resulted in inactivation with the loss of one sulfhydryl group/enzyme subunit. Of 4 cysteine residues in the Rhodococcus nitrilase, only Cys-165 is conserved in the Klebsiella nitrilase. Mutant enzymes containing Ala or Ser instead of Cys-165 did not exhibit nitrilase activity. These findings suggest that Cys-165 plays an essential role in the function of the active site.     

Fluorescence energy transfer between cysteine 199 and cysteine 343: evidence for MgATP-dependent conformational change in the catalytic subunit of cAMP-dependent protein kinase

The catalytic subunit of cAMP-dependent protein kinase has two cysteine residues, Cys 199 and Cys 343, which are protected against alkylation by MgATP [Nelson, N. C., & Taylor, S. S. (1981) J. Biol. Chem. 256, 3743]. While Cys 199 is in close proximity to the active site of the catalytic subunit and is probably directly protected against alkylation by MgATP, the mechanism by which MgATP prevents alkylation of Cys 343 is unclear. To determine whether MgATP directly protects Cys 343 from alkylation by being in close proximity to both Cys 199 and the MgATP binding site, fluorescence resonance energy transfer techniques were used to measure the distance between Cys 199 and Cys 343. Two different donor-acceptor pairs containing 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole at Cys 199 as the acceptor and either 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2, 8- dione or N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine at Cys 343 as the donor were prepared following the method described in the preceding paper [First, E. A., & Taylor, S. S. (1989) Biochemistry (preceding paper in this issue)]. From the efficiencies of fluorescence resonance energy transfer for each donor-acceptor pair, the distance between Cys 199 and Cys 343 was estimated to be between 31 and 52 A. Since Cys 199 is close to the MgATP binding site and since MgATP cannot extend beyond a distance of 16 A, it is unlikely that Cys 343 at a distance of at least 31 A from Cys 199 is in direct contact with the bound nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni

Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.     

Selective modification of the catalytic subunit of cAMP-dependent protein kinase with sulfhydryl-specific fluorescent probes

The catalytic subunit of cAMP-dependent protein kinase contains only two cysteine residues, and the side chains of both Cys 199 and Cys 343 are accessible. Modification of the catalytic subunit by a variety of sulfhydryl-specific reagents leads to the loss of enzymatic activity. The differential reactivity of the two sulfhydryl groups at pH 6.5 has been utilized to selectively modify each cysteine with the following fluorescent probes: 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene- 2,8-dione, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, and 4-[N-[(iodoacetoxy)ethyl]-N-methyl-amino]-7-nitrobenz-2-oxa-1,3-diazole. The most reactive cysteine is Cys 199, and exclusive modification of this residue was achieved with each reagent at pH 6.5. Modification of Cys 343 required reversible blocking of Cys 199 with 5,5'-dithiobis(2-nitrobenzoic acid) followed by reaction of Cys 343 with the fluorescent probe at pH 8.3. Treatment of this modified catalytic subunit with reducing reagent restored catalytic activity by unblocking Cys 199. In contrast, catalytic subunit that was selectively labeled at Cys 199 by the fluorescent probes was catalytically inactive. Even though Cys 199 is presumably close to the interaction site between the regulatory subunit and the catalytic subunit, all of the modified C-subunits retained the capacity to aggregate with the type II regulatory subunit in the absence of cAMP, and the resulting holoenzymes were dissociated in the presence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the mechanism of action of histone kinase dependent on adenosine 3':5'-monophosphate. Evidence for involvement of histidine and lysine residues in the phosphotransferase reaction.

The reaction of the phosphate residue transfer catalysed by histone kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) was studied. The phosphotransferase reaction was shown to obey the mechanism of ping-pong bi-bi type. After incubation of the catalytic subunit of histone kinase with [gamma-32P]ATP the incorporation of one mole of [32P]phosphage per mole of protein was observed. The tryptic [32P]phosphohistidine-containing peptide was isolated and its N-terminus and amino acid composition were determined. The 2',3'-dialdehyde derivative of ATP (oATP) was used as the affinity label for the catalytic subunit of cyclic-AMP-dependent histone kinase. The inhibitor formed an alidmine bond with epsilon-amino group of the lysine residue of the active site and was irreversibly bound to the enzyme after reduction by sodium borohydride with concurrent irreversible inactivation of the enzyme. After inactivation, about one mole of 14C-labelled inhibitor was incorporated per mole of the enzyme. ATP effectively protected the catalytic subunit of histone kinase against inactivation by oATP. Tryptic digestion of the enzyme-inhibitor complex led to the isolation of the 14C-labelled peptide of the active site of histone kinase. Basing on these results, the role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.     

The cDNA sequence of the 69-kDa subunit of the carrot vacuolar H+-ATPase. Homology to the beta-chain of F0F1-ATPases

Vacuolar ATPases constitute a novel class of N-ethylmaleimide- and nitrate-sensitive proton pumps associated with the endomembrane system of eukaryotic cells. They resemble F0F1-ATPases in that they are large multimeric proteins, 400-500 kDa, composed of three to nine different subunits. Previous studies have indicated that the active site is located on the approximately 70-kDa subunit. Using antibodies to the approximately 70-kDa subunit of corn to screen a carrot root lambda gt11 cDNA library, we have isolated cDNA clones of the carrot 69-kDa subunit. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA. The 69-kDa subunit consists of 623 amino acids (Mr 68,835), with no obvious membrane-spanning regions. The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone. The protein sequence of the carrot 69-kDa subunit also exhibited 34.3% identity to four representative F0F1-ATPase beta-chains over a 275-amino-acid core stretch of similar sequence. Alignment studies revealed several regions which were highly homologous to beta-chains, including sequences previously implicated in catalytic function. This provides definitive evidence that the vacuolar ATPase is closely related to the F0F1-type ATPases. A major functional difference between the 69-kDa and beta-subunits is the location of 3 critical cysteine residues: two in the putative catalytic region (Cys-248 and Cys-256) and one in the proposed Mg2+-binding site (Cys-279). These cysteines (and two others) probably account for the sensitivity of the vacuolar H+-ATPase to the sulfhydryl reagent, N-ethylmaleimide. It is proposed that the two ATPases may have arisen from a common ancestor by the insertion or deletion of a large stretch of nonhomologous sequence near the amino-terminal end of the subunit.     

Location and mechanism of alpha 2,6-sialyltransferase dimer formation. Role of cysteine residues in enzyme dimerization, localization, activity, and processing

A significant proportion of the alpha2,6-sialyltransferase of protein Asn-linked glycosylation (ST6Gal I) forms disulfide-bonded dimers that exhibit decreased activity, but retain the ability to bind asialoglycoprotein substrates. Here, we have investigated the subcellular location and mechanism of ST6Gal I dimer formation, as well as the role of Cys residues in the enzyme's trafficking, localization, and catalytic activity. Pulse-chase analysis demonstrated that the ST6Gal I disulfide-bonded dimer forms in the endoplasmic reticulum. Mutagenesis experiments showed that Cys-24 in the transmembrane region is required for dimerization, while catalytic domain Cys residues are required for trafficking and catalytic activity. Replacement of Cys-181 and Cys-332 generated proteins that are largely retained in the endoplasmic reticulum and minimally active or inactive, respectively. Replacement of Cys-350 or Cys-361 inactivated the enzyme without compromising its localization or processing, suggesting that these amino acids are part of the enzyme's active site. Replacement of Cys-139 or Cys-403 generated proteins that are catalytically active and appear to be more stably localized in the Golgi, since they exhibited decreased cleavage and secretion. The Cys-139 mutant also exhibited increased dimer formation suggesting that ST6Gal I dimers may be critical in the oligomerization process involved in stable ST6Gal I Golgi localization.     

Contribution of the Disulfide Bond of the A Subunit to the Action of Escherichia coli Heat-Labile Enterotoxin

Escherichia coli heat-labile enterotoxin (LT) consists of an A subunit and five B subunits. These subunits oligomerize into an assembled holotoxin within the periplasm. Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus. This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm. However, it is not known whether other regions of the A subunit contribute to the assembly. The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199. It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein. We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit. We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes. The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly. A reconstitution experiment in vitro supported the notion. Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs. These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.     

Subunit complementation of thymidylate synthase.

Each of the two active sites of thymidylate synthase contains amino acid residues contributed by the other subunit. For example, Arg-178 of one monomer binds the phosphate group of the substrate dUMP in the active site of the other monomer [Hardy et al. (1987) Science 235, 448-455]. Inactive mutants of such residues should combine with subunits of other inactive mutants to form heterodimeric hybrids with one functional active site. In vivo and in vitro approaches were used to test this hypothesis. In vivo complementation was accomplished by cotransforming plasmid mixtures encoding pools of inactive Arg-178 mutants and pools of inactive Cys-198 mutants into a host strain deficient in thymidylate synthase. Individual inactive mutants of Arg-178 were also cotransformed with the C198A mutant. Subunit complementation was detected by selection or screening for transformants which grew in the absence of thymidine, and hence produced active enzyme. Many mutants at each position representing a wide variety of size and charge supported subunit complementation. In vitro complementation was accomplished by reversible dissociation and unfolding of mixtures of purified individual inactive Arg-178 and Cys-198 mutant proteins. With the R178F + C198A heterodimer, the Km values for dUMP and CH2H4folate were similar to those of the wild-type enzyme. By titrating C198A with R178F under unfolding-refolding conditions, we were able to calculate the kcat value for the active heterodimer. The catalytic efficiency of the single wild-type active site of the C198A + R178F heterodimer approaches that of the wild-type enzyme.     

Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase

The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS). An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data. The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations. Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide. The protein contains no free sulfhydryl groups. The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319. In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins. The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface. A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance. In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal. Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.     

Mechanism of activation of cAMP-dependent protein kinase: in Mucor rouxii the apparent specific activity of the cAMP-activated holoenzyme is different than that of its free catalytic subunit

Kinetic constants for peptide phosphorylation by the catalytic subunit of the dimorphic fungus Mucor rouxii protein kinase A were determined using 13 peptides derived from the peptide containing the basic consensus sequence RRASVA, plus kemptide, S6 peptide, and protamine. As a whole, although with a greater Km, the order of preference of the peptides by the M. rouxii catalytic subunit was similar to the one displayed by mammalian protein kinase A. Particularly significant is the replacement of serine by threonine in the basic peptide RRATVA, which impaired its role as a substrate of M. rouxii catalytic subunit. Mucor rouxii protein kinase A is a good model in which to study the mechanism of activation since cAMP alone is not enough to promote activation and dissociation. Four peptides were selected for the study of holoenzyme activation under conditions in which the enzymatic activity was not proportional to the holoenzyme concentration: RRASVA, RRRRASVA, KRRRLSSRA (S6 peptide), and LRRASLG (kemptide); protamine was used as reference. Differential activation degree was observed depending on the peptide used and on cAMP concentration. Ratios of activity between different substrates displayed by the holoenzyme under the above conditions did not reflect the one expected for the free catalytic subunit. The degree of inhibition of the holoenzyme activity by an active peptide derived from the thermostable protein kinase inhibitor was dependent on the substrate used and on the holoenzyme concentration, while it was found to be independent of these two parameters for free catalytic subunit. Polycation modulation of holoenzyme activation by cAMP was also dependent on the polycation itself and on the peptide used as substrate. The observed kinetic differences between holoenzyme and free catalytic subunit were decreased or almost abolished when working at low enzyme or at high cAMP concentrations. Two hypotheses compatible with the results are discussed: substrate participation in the dissociation process and/or holoenzyme activation without dissociation.