Functional role of cysteinyl residues in tryptophanase

Holotryptophanase inactivated by oxidation of cysteinyl residues showed a different absorption spectrum from the native enzyme. At pH 8.0, the native enzyme preferentially existed as a 337-nm species (active form), whereas in the inactive enzyme a 420-nm species (inactive form) was dominant. During the reactivation of the enzyme by reduction with dithiothreitol, an increase at 337 nm and a decrease at 420 nm were observed with concomitant increase in enzymatic activity, which was accompanied by the appearance of two cysteinyl residues per monomer. Specific S-cyanylation of cysteinyl residues by nitrothiocyanobenzoic-acid-inactivated apotryptophanase with the modification of one cysteinyl residue per monomer, whereas holotryptophanase was highly resistant to inactivation with nitrothiocyanobenzoic acid. The essential role of the active-site-bound pyridoxal 5'-phosphate in protection against inactivation was confirmed by the agreement of the K1/2 (protection) of 5.0 microM for pyridoxal 5'-phosphate with Km of 2.0 microM in enzyme catalysis. The inactivation by nitrothiocyanobenzoic acid caused a similar shift in the equilibrium between the 337-nm species and 420-nm species, i.e. decrease of the 337-nm species and increase of the 420-nm species. From the pH dependence of the equilibrium between these two species, pKa of 7.9 and 7.4 was obtained for the inactive and the dithiothreitol-activated enzyme, respectively, indicating that cysteinyl residue(s) participated in lowering the pKa of the interconversion between the 337-nm species (active form) and 420-nm species (inactive form). The possible role of cysteinyl residues in the function of tryptophanase is discussed.     

The functional role of cysteine residues for c-Abl kinase activity

S-glutathionylation, the formation of mixed disulfides of glutathione with cysteine residues of proteins, is a broadly observed physiological modification that occurs in response to oxidative stress. Since cysteine residues are particularly susceptible to oxidative modification by reactive oxygen species, S-glutathionylation can protect proteins from irreversible oxidation. In this study, we show that the kinase activity of the non-receptor tyrosine kinase c-Abl is inhibited by in vitro thiol modification; specifically, the cysteine residues of c-Abl are modified by S-glutathionylation and by thiol alkylating agents such as 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid and N-ethylmaleimide. Modification of cysteine residues of c-Abl tyrosine kinase using glutathione disulfide and thiol alkylating agents corresponds to a concomitant loss of kinase activity. We also demonstrate that S-glutathionylation of c-Abl can be reversed using a physiological system involving glutaredoxin and this reversal restores c-Abl kinase activity. To our knowledge, these are the first data to show S-glutathionylation of c-Abl, and this modification may represent a mechanism of regulation of c-Abl kinase activity in cells under oxidative stress.     

The phosphorylase kinase activity of hearts from phosphorylase kinase-deficient mice

In an assay measuring radioactive incorporation from gamma--P32P]ATP into phosphorylase b, cardiac muscle extracts from mice with the phosphorylase kinase deficiency mutation showed significant, calcium-dependent phosphorylase kinase activity that was 10 to 15% of that of Swiss mice, the control strain. Isoproterenol stimulated significant phosphorylase a accumulation in both isolated atria and right ventricular strips of phosphorylase kinase-deficient mice, and the drug-stimulated increases in phosphorylase a activity the the contractile responses of right ventricular strips were similar in Swiss and phosphorylase kinase/deficient mice.     

Inactivation of purine nucleoside phosphorylase by modification of arginine residues.

Treatment of purine nucleoside phosphorylase (EC 2.4.2.1), from either calf spleen or human erythrocytes, with 2,3-butanedione in borate buffer or with phenylglyoxal in Tris buffer markedly decreased the enzyme activity. At pH 8.0 in 60 min, 95% of the catalytic activity was destroyed upon treatment with 33 mM phenylglyoxal and 62% of the activity was lost with 33 mm 2,3-butanedione. Inorganic phosphate, ribose-1-phosphate, arsenate, and inosine when added prior to chemical modification all afforded protection from inactivation. No apparent decrease in enzyme catalytic activity was observed upon treatment with maleic anhydride, a lysine-specific reagent. Inactivation of electrophoretically homogeneous calf-spleen purine nucleoside phosphorylase by butanedione was accompanied by loss of arginine residues and of no other amino acid residues. A statistical analysis of the inactivation data vis-à-vis the fraction of arginines modified suggested that one essential arginine residue was being modified.     

The role of phosphorylase kinase subunits in interaction with glycogen

The interaction of rabbit skeletal muscle phosphorylase kinase with CNBr-activated glycogen results in the formation of a covalent complex. The non-bound kinase was removed by chromatography on DEAE-cellulose and phenyl-Sepharose. The amount of the bound protein increased with an increase in the number of activated groups in the glycogen molecule; the enzyme activity was thereby decreased. The kinase covalently and non-covalently bound to glycogen exhibited a higher affinity for the protein substrate (phosphorylase b) as well as for Mg2+ and Ca2+ than did the kinase in the absence of glycogen. Electrophoresis performed under denaturating conditions showed that the gamma-subunit of phosphorylase kinase is responsible for the enzyme binding to CNBr-glycogen. The effect of cross-linking reagents (glutaric aldehyde, 1.5-difluoro-2.4-dinitrobenzene) on the binding of phosphorylase kinase subunits was studied. Glycogen afforded protection of the gamma-subunit from the cross-linking to other enzyme subunits. An analysis of the subunit composition of phosphorylase kinase covalently bound to CNBr-glycogen and of the enzyme treated with cross-linking reagents in the presence of glycogen-revealed that the gamma-subunit is involved in the specific binding of phosphorylase kinase to glycogen.     

The control of phosphorylase kinase phosphatase activity by polycations and the deinhibitor protein

The dephosphorylation of phosphorylase beta kinase by the activated ATP, Mg-dependent protein phosphatase, which is highly specific for the beta-subunit, is stimulated by the deinhibitor protein which neutralizes the effect of inhibitor-1 and the modulator protein on the phosphatase. The specific dephosphorylation of the alpha-subunit of phosphorylase beta kinase by a "latent" protein phosphatase isolated from vascular smooth muscle is stimulated by histone H1 but not affected by the deinhibitor protein. These observations show that there is no strict correlation between the insensitivity of a protein phosphatase to inhibitor-1 or modulator protein and the dephosphorylation of the alpha-subunit of phosphorylase beta kinase.     

Structural and functional role of cysteinyl residues in tobacco acetolactate synthase

Acetolactate synthase (ALS) is the common enzyme in the biosynthesis of valine, leucine, and isoleucine. The role of four cysteinyl residues in tobacco ALS was determined using site-directed mutagenesis and cysteine-specific cleavage. The C411A mutation abolished the enzymatic activity, as well as the binding affinity for the cofactor FAD. The activation constant of C411S for FAD is approximately 50-fold higher than that of wALS. The C607S mutation did not significantly affect the kinetic parameters. The IC(50) values of C411S and C607S for ALS-inhibiting herbicides are not much different from those of wALS. Two mutants, C163S and C309S, are labile and readily degraded to peptide fragments. The treatment of wALS with 2-nitro-5-thiocyanobenzoic acid, specific for cleavage of the N-terminal side of cysteine, yielded three peptides of 37.0, 22. 0, and 7.0 kDa. This fragmentation pattern is consistent with that deduced from the amino acid sequence of tobacco ALS, assuming the disulfide bond between Cys163 and Cys309. These results suggest that Cys411 is involved in the binding of FAD and that the intrachain disulfide bond between Cys163 and Cys309 plays a key role in maintaining the correct conformation of tobacco ALS.     

Roles of cysteinyl residues of phosphoribulokinase as examined by site-directed mutagenesis.

The Calvin Cycle enzyme phosphoribulokinase is activated in higher plants by the reversible reduction of a disulfide bond, which is located at the active site. To determine the possible contribution of the two regulatory residues (Cys16 and Cys55) to catalysis, site-directed mutagenesis has been used to replace each of them in the spinach enzyme with serine or alanine. The only other cysteinyl residues of the kinase, Cys244 and Cys250, were also replaced individually by serine or alanine. A comparison of specific activities of native and mutant enzymes reveals that substitutions at positions 244 or 250 are inconsequential. The position 16 mutants retain 45-90% of the wild-type activity and display normal Km values for both ATP and ribulose 5-phosphate. In contrast, substitution at position 55 results in 85-95% loss of wild-type activity, with less than a 2-fold increase in the Km for ATP and a 4-8-fold increase in the Km for ribulose 5-phosphate. These results are consistent with moderate facilitation of catalysis by Cys55 and demonstrate that the other three cysteinyl residues do not contribute significantly either to structure or catalysis. The enhanced stability, relative to wild-type enzyme, of the Ser16 mutant protein to a sulfhydryl reagent supports earlier suggestions that Cys16 is the initial target of the oxidative deactivation process.     

The ATPase activity of phosphorylase kinase is regulated in parallel with its protein kinase activity.

Phosphorylase kinase from rabbit skeletal muscle has been found to have an intrinsic ATPase activity that occurs at a rate approximately 0.2% of that of its phosphorylase conversion activity and about three times that of its autophosphorylation activity. The characteristics of this ATPase activity were in all aspects tested essentially the same as the kinase's phosphorylase conversion activity. The ATPase requires Mg2+ and is dramatically stimulated by Ca2+ ions. At neutral pH there is a pronounced lag in the rate of product formation that is not present at alkaline pH, a condition that greatly stimulates both the phosphorylase conversion and ATPase activities. ATP is preferentially hydrolyzed over GTP and the Km for MgATP determined in the ATPase assay is 0.14 mM. ADP, an allosteric activator of phosphorylase conversion, also stimulates the ATPase activity, whereas beta-glycerophosphate, an inhibitor of phosphorylase conversion, is an inhibitor of the ATPase activity. Phosphorylation or partial proteolysis of the kinase, which are known to activate phosphorylase conversion, also activate the ATPase activity. Because the phosphorylase conversion and ATPase activities are regulated in parallel, we conclude that activation of the two catalytic activities must share a common underlying basis, namely an enhanced phosphotransferase activity that is independent of the phosphoryl acceptor.     

pCMB treatment reveals the essential role of cysteinyl residues in conferring functional competence to the regulatory subunit of protein kinase CK2

To assess the functional role of the four conserved cysteinyl residues in the regulatory beta-subunit of protein kinase CK2, the effect of pCMB and other reagents of sulfhydryl groups has been investigated. The pCMB-treated beta-subunit has lost its ability to form either homodimers or regular alpha(2)beta(2) heterotetramers with the catalytic subunit. It also fails to increase catalytic activity toward peptide substrates and to mediate the stimulatory effect of polylysine. The pCMB-treated beta-subunit, however, is still able to prevent calmodulin phosphorylation and to physically interact with the alpha-subunit to form inactive complexes whose sedimentation coefficient is lower than that of CK2 holoenzyme. These inactive complexes upon treatment with reducing agents like DTT are converted into a fully active heterotetrameric holoenzyme.     

Block of sodium channels by divalent mercury: role of specific cysteinyl residues in the P-loop region

Divalent mercury (Hg(2+)) blocked human skeletal Na(+) channels (hSkM1) in a stable dose-dependent manner (K(d) = 0.96 microM) in the absence of reducing agent. Dithiothreitol (DTT) significantly prevented Hg(2+) block of hSkM1, and Hg(2+) block was also readily reversed by DTT. Both thimerosal and 2,2'-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg(2+) block of hSkM1. Y401C+E758C rat skeletal muscle Na(+) channels (mu1) that form a disulfide bond spontaneously between two cysteines at the 401 and 758 positions showed a significantly lower sensitivity to Hg(2+) (K(d) = 18 microM). However, Y401C+E758C mu1 after reduction with DTT had a significantly higher sensitivity to Hg(2+) (K(d) = 0.36 microM) than wild-type hSkM1. Mutants C753Amu1 (K(d) = 8.47 microM) or C1521A mu1 (K(d) = 8.63 microM) exhibited significantly lower sensitivity to Hg(2+) than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region may play an important role in the Hg(2+) block of the hSkM1 isoform. The heart Na(+) channel (hH1) was significantly more sensitive to low-dose Hg(2+) (K(d) = 0.43 microM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance (K(d) = 1.12 microM) to Hg(2+) than did wild-type hH1. In summary, Hg(2+) probably inhibits the muscle Na(+) channels at more than one cysteinyl residue in the Na(+) channel P-loop region. Hg(2+) exhibits a lower K(d) value (<1. 23 microM) for inhibition by forming a sulfur-Hg-sulfur bridge, as compared to reaction at a single cysteinyl residue with a higher K(d) value (>8.47 microM) by forming sulfur-Hg(+) covalently. The heart Na(+) channel isoform with more than two cysteinyl residues in the P-loop region exhibits an extremely high sensitivity (K(d) < 0. 43 microM) to Hg(+), accounting for heart-specific high sensitivity to the divalent mercury.     

Thiophosphate-activated phosphorylase kinase as a probe in the regulation of phosphorylase phosphatase.

Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.     

Regulation of the activity of rabbit skeletal muscle phosphorylase kinase by glycogen

The effects of glycogen on the non-activated and activated forms of phosphorylase kinase were studied. It was found that in the presence of glycogen the activity of non-activated kinase at pH 6.8 and 8.2 and that of the activated (in the course of phosphorylation) form are enhanced. The degree of activation depends on glycogen concentration. At saturating concentrations, this enzyme activity increases 2-3-fold; the enzyme affinity for the protein substrate, phosphorylase b, also shows an increase. The polysaccharide has no effect on the activity of phosphorylase kinase stimulated by limited proteolysis. In the presence of glycogen, the rate of autocatalytic phosphorylation of the enzyme is increased. Glycogen stabilizes the enzyme activity upon dilution. The experimental results suggest that the polysaccharide directly affects the phosphorylase kinase molecule. The maximal binding was shown to occur at the enzyme/polysaccharide ratio of 1:10 (w/w) in the presence of Ca2+ and Mg2+.     

Motility of the N-terminal tail of phosphorylase b as revealed by crosslinking.

There are lysyl-ε-NH₂ groups within about 3.5 Å distance across the intersubunit contact area of rabbit muscle phosphorylase $\text{b}$, as shown by cross-linking with malonic diimidate. These include the lysines of N-terminal region as revealed by limited tryptic digestion, but the contribution of the tail lysines to overall formation of covalent dimers is small. The fine structure of dimer band on dodecylsulfate-gelelectrophoretograms of crosslinked phosphorylases suggests that the tail retains its freedom in the phosphorylase $\text{b}$-AMP complex. Amidination induces the dissociation of phosphorylase $\text{b}$ dimer, which is slow relative to crosslinking.     

Chemical modification of rabbit skeletal muscle phosphorylase kinase with phenylglyoxal

Nonactivated phosphorylase kinase from rabbit skeletal muscle is inactivated by treatment with phenylglyoxal. Under mild reaction conditions, a derivative that retains 10-15% of the pH 8.2 catalytic activity is obtained. The kinetics of inactivation profile, differential effects of modification on pH 6.8 and 8.2 catalytic activities, and the insensitiveness of the modified enzyme to activation by ADP reveal that the 10-15% of catalytic activity remaining is very likely due to intrinsic catalytic activity of the derivative rather than to the presence of unmodified enzyme molecules. The kinetic results also suggest that the inactivation is correlatable with the reaction of one molecule of the reagent with the enzyme without any prior binding of phenylglyoxal. The phenylglyoxal modification reduces the autophosphorylation rate of the kinase. Autophosphorylated phosphorylase kinase is inactivated by phenylglyoxal at a much slower rate than the inactivation of nonactivated kinase. Thus, phenylglyoxal modification influences the phosphorylation and vice versa. The modified enzyme can be reactivated by treatment with trypsin or by dissociation using chatropic salts. The activity of the phenylglyoxal-modified enzyme after trypsin digestion or dissociation with LiBr reaches the same level as that of the native enzyme digested with trypsin or treated with LiBr under identical conditions. The results suggest that the effect of modification is overcome by dissociation of the subunits of phosphorylase kinase and that the catalytic site is not modified under conditions when 85% of the pH 8.2 catalytic activity is lost. Among various nucleotides and metal ions tested, only ADP, with or without Mg2+, afforded effective protection against inactivation with phenylglyoxal. At pH 6.8, 1 mM ADP afforded complete protection against inactivation. Experiments with 14C-labeled phenylglyoxal revealed that ADP seemingly protects one residue from modification. This result is in agreement with the kinetic result that the inactivation seemingly is due to reaction of one molecule of the reagent with the enzyme. The results confirm the existence of a high-affinity ADP binding site on nonactivated phosphorylase kinase and suggest the involvement of a functional arginyl residue at or near the ADP binding site in the regulation of of pH 8.2 catalytic activity of the enzyme.     

The elusive activity of the Yersinia protein kinase A kinase domain is revealed

Yersinia spp. pathogens use their type III secretion system to translocate effectors that manipulate host signaling pathways during infection. Although molecular targets for five of the six known Yersinia effectors are known, the target for the serine/threonine kinase domain of Yersinia protein kinase A (YpkA) has remained elusive. Recently, Navarro et al. (2007) demonstrated that YpkA phosphorylates Galphaq, and inhibits Galphaq-mediated signaling. Inhibition by YpkA could contribute to one of the most documented symptoms of Yersinia pestis infection, extensive bleeding.